Docosahexaenoic acid producing strains of yarrowia lipolytica

ABSTRACT

An engineered strain of the oleaginous yeast  Yarrowia lipolytica  capable of producing greater than 5.6% docosahexaenoic acid acid (DHA, an w-3 polyunsaturated fatty acid) in the total oil fraction is described. This strain comprises various chimeric genes expressing heterologous desaturases, elongases and acyltransferases and optionally comprises various native desaturase and acyltransferase knockouts to enable synthesis and high accumulation of DHA. Production host cells are claimed, as are methods for producing DHA within said host cells.

This application claims the benefit of U.S. Provisional Application No.60/624,812, filed Nov. 4, 2004, the entire contents of which are hereinincorporated by reference.

FIELD OF THE INVENTION

This invention is in the field of biotechnology. More specifically, thisinvention pertains to an engineered strain of the oleaginous yeastYarrowia lipolytica that is capable of producing docosahexaenoic acid(an ω-3 polyunsaturated fatty acid) in high concentrations.

BACKGROUND OF THE INVENTION

Docosahexaenoic acid (DHA; cis-4, 7, 10, 13, 16, 19-docosahexaenoicacid; C22:6 ω-3) is essential for the growth, functional development andhealthy maintenance of brain function and is required throughout lifefrom infancy through aging (Horrocks, L. A. and Y. K. Yeo. Pharmacol.Res. 40(3):211-225 (1999)). DHA deficiencies are associated with foetalalcohol syndrome, attention deficit hyperactivity disorder, cysticfibrosis, phenylketonuria, unipolar depression, aggressive hostility andadrenoleukodystrophy. In contrast, increased intake of DHA has beenshown to be beneficial or have a positive effect in inflammatorydisorders (e.g., rheumatoid arthritis), Type II diabetes, hypertension,atherosclerosis, depression, myocardial infarction, thrombosis, somecancers and for prevention of the onset of degenerative disorders suchas Alzheimer's disease.

Fish (e.g., salmon, trout, mackerel) are an important source of DHA,since they naturally contain high concentrations of this long-chainfatty acid. Based on abundant research [reviewed in the 2005 DietaryGuidelines Advisory Committee Report for Americans, part D, section 4(coordinated by the U.S. Dept. of Health & Human Services and the U.S.Dept. of Agriculture)], the American Heart Association, the NationalCholesterol Education Program, the World Health Association, theEuropean Society for Cardiology, the American Diabetes Association andthe United Kingdom Scientific Advisory Committee on Nutrition allrecommend two servings of fish per week (wherein each serving providesthe equivalent of ˜450 mg per day of DHA and eicosapentaenoic acid (EPA,C20:5 ω-3)) for the cardioprotective effects so conveyed. As such, DHAis incorporated into a variety of products relating to functional foods,infant nutrition, bulk nutrition and animal health.

Although the physiological functions of docosapentaenoic acid (DPA,C22:5 ω-3) are still unknown, this fatty acid is the metabolic precursorof DHA and an immediate down-stream product of EPA via elongation. DPAis also known to be contained in fish oil, although the content isextremely low. The only known function for DPA is its usefulness as acarrier for transporting pharmaceutical agents into the brain (JapanesePatent Publication (Kokai) No. 61-204136 (1986)). It is expected,however, that DPA may play a physiological role in the animal body,since it is known that DPA increases in compensation for a lack of DHA(Homayoun et al., J. Neurochem., 51:45 (1988); Hamm et al., Biochem. J.,245:907 (1987); and Rebhung et al., Biosci. Biotech. Biochem., 58:314(1994)). Thus, both DPA and DHA must be considered as important ω-3fatty acids. One skilled in the art will recognize that the teachingsherein that are directed toward DHA are also largely applicable andrelevant to DPA production, should that become a desirable product inthe future.

Although DHA is naturally found in different types of fish oil andmarine plankton, it is expected that the supply of this ω-3 fatty acidwill not be sufficient to meet growing demands. Fish oils have highlyheterogeneous compositions (thereby requiring extensive purification toenrich for DHA), unpleasant tastes and odors (making removaleconomically difficult and rendering the oils unacceptable as foodingredients), and are subject to environmental bioaccumulation of heavymetal contaminants and fluctuations in availability (due to weather,disease and/or over-fishing).

As an alternative to fish oil, DHA can also be produced microbially.Generally, microbial oil production involves cultivating an appropriatemicroorganism that is naturally capable of synthesizing DHA in asuitable culture medium to allow for oil synthesis (which occurs in theordinary course of cellular metabolism), followed by separation of themicroorganism from the fermentation medium and treatment for recovery ofthe intracellular oil. Numerous different processes exist based on thespecific microbial organism utilized [e.g., Schizochytrium species (U.S.Pat. No. 5,340,742; U.S. Pat. No. 6,582,941); Ulkenia (U.S. Pat. No.6,509,178); Pseudomonas sp. YS-180 (U.S. Pat. No. 6,207,441);Thraustochytrium genus strain LFF1 (U.S. 2004/0161831 A1);Crypthecodinium cohnii (U.S. 2004/0072330 A1; de Swaaf, M. E. et al.Biotechnol Bioeng. 81(6):666-72 (2003) and Appl Microbiol Biotechnol.61(1):40-3 (2003)); Emiliania sp. (Japanese Patent Publication (Kokai)No. 5-308978 (1993)); and Japonochytrium sp. (ATCC #28207; JapanesePatent Publication (Kokai) No.199588/1989)]. Additionally, the followingmicroorganisms are known to have the ability to produce DHA: Vibriomarinus (a bacterium isolated from the deep sea; ATCC #15381); themicro-algae Cyclotella cryptica and Isochrysis galbana; and, flagellatefungi such as Thraustochytrium aureum (ATCC #34304; Kendrick, Lipids,27:15 (1992)) and the Thraustochytrium sp. designated as ATCC #28211,ATCC #20890 and ATCC #20891. And, although several of these processesare not adaptable for industrial commercialization as a result ofvarious limitations, there are at least three different fermentationprocesses for commercial production of DHA: fermentation of C. cohniifor production of DHASCO™ (Martek Biosciences Corporation, Columbia,Md.); fermentation of Schizochytrium sp. for production of an oilformerly known as DHAGold (Martek Biosciences Corporation); andfermentation of Ulkenia sp. for production of DHActive™ (Nutrinova,Frankfurt, Germany)). Despite these successes, each of these methodssuffer from an inability to substantially improve the yield of oil or tocontrol the characteristics of the oil composition produced, since thefermentations rely on the natural abilities of the microbes themselves.

Thus, microbial production of DHA using recombinant means is expected tohave several advantages over production from natural microbial sources.For example, recombinant microbes having preferred characteristics foroil production can be used, since the naturally occurring microbialfatty acid profile of the host can be altered by the introduction of newbiosynthetic pathways in the host and/or by the suppression of undesiredpathways, thereby resulting in increased levels of production of desiredPUFAs (or conjugated forms thereof) and decreased production ofundesired PUFAs. Secondly, recombinant microbes can provide PUFAs inparticular forms which may have specific uses. And, finally, microbialoil production can be manipulated by controlling culture conditions,notably by providing particular substrate sources for microbiallyexpressed enzymes, or by addition of compounds/genetic engineering tosuppress undesired biochemical pathways. Thus, for example, it ispossible to modify the ratio of ω-3 to ω-6 fatty acids so produced, orengineer production of a specific PUFA (e.g., DHA) without significantaccumulation of other PUFA downstream or upstream products.

Microbial production of DHA first requires the synthesis of theintermediate fatty acid, EPA. And, most microbially produced DHA issynthesized via the Δ6 desaturase/elongase pathway (which ispredominantly found in higher plants, algae, mosses, fungi, nematodesand humans) and wherein: 1.) oleic acid is converted to LA by the actionof a Δ12 desaturase; 2.) optionally, LA is converted to ALA by theaction of a Δ15 desaturase; 3.) LA is converted to GLA, and/or ALA isconverted to STA, by the action of a Δ6 desaturase; 3.) GLA is convertedto DGLA, and/or STA is converted to ETA, by the action of a C_(18/20)elongase; 3.) DGLA is converted to ARA, and/or ETA is converted to EPA,by the action of a Δ5 desaturase; and 4.) optionally, ARA is convertedto EPA by the action of a Δ17 desaturase (FIG. 1). However, an alternateΔ9 elongase/Δ8 desaturase pathway for the biosynthesis of EPA operatesin some organisms, such as euglenoid species, where it is the dominantpathway for formation of C₂₀ PUFAs (Wallis, J. G., and Browse, J. Arch.Biochem. Biophys. 365:307-316 (1999); WO 00/34439; and Qi, B. et al.FEBS Letters. 510:159-165 (2002)). In this pathway, 1.) LA and ALA areconverted to EDA and ETrA, respectively, by a Δ9 elongase; 2.) EDA andETrA are converted to DGLA and ETA, respectively, by a Δ8 desaturase;and 3.) DGLA and ETA are ultimately converted to EPA, as describedabove. Upon synthesis of EPA, a C_(20/22) elongase is responsible forconversion of the substrate to DPA, followed by desaturation by a Δ4desaturase to yield DHA.

The literature reports a number of recent examples whereby variousportions of the ω-3/ω-6 PUFA biosynthetic pathway have been introducedinto Saccharomyces cerevisiae (a non-oleaginous yeast). Specifically,Dyer, J. M. et al. (Appl. Eniv. Microbiol., 59:224-230 (2002)) reportedsynthesis of linolenic acids; Knutzon et al. (U.S. Pat. No. 6,136,574)demonstrated production of linoleic acid (LA), γ-linolenic acid (GLA),ALA and stearidonic acid (STA); Domergue, F. et al. (Eur. J. Biochem.269:4105-4113 (2002)) described production of EPA; and Pereira, S. L. etal. (Biochem. J. 384:357-366 (2004)) were the first to produce DHA (3.8%of the total fatty acids, when fed EPA as substrate). Despite thesesuccesses, however, complex metabolic engineering has not been reportedthat would enable economical production of commercial quantities of DHA(i.e., greater than 5-30% with respect to total fatty acids).Additionally, considerable discrepancy exists concerning the mostappropriate choice of host organism for such engineering.

Recently, Picataggio et al. (WO 2004/101757 and co-pending U.S. PatentApplication No. 60/624,812) have explored the utility of oleaginousyeast, and specifically, Yarrowia lipolytica (formerly classified asCandida lipolytica), as a preferred class of microorganisms forproduction of PUFAs such as ARA, EPA and DHA. Oleaginous yeast aredefined as those yeast that are naturally capable of oil synthesis andaccumulation, wherein oil accumulation can be up to about 80% of thecellular dry weight. Despite a natural deficiency in the production ofω-6 and ω-3 fatty acids in these organisms (since naturally producedPUFAs are limited to 18:2 fatty acids (and less commonly, 18:3 fattyacids)), Picataggio et al. (supra) have demonstrated production of 1.3%ARA and 1.9% EPA (of total fatty acids) in Y. lipolytica usingrelatively simple genetic engineering approaches and up to 28% EPA usingmore complex metabolic engineering. However, similar work has not beenperformed to enable economic, commercial production of DHA in thisparticular host organism.

Applicants have solved the stated problem by engineering various strainsof Yarrowia lipolytica that are capable of producing greater than 5% DHAin the total oil fraction, using the Δ6 desaturase/Δ6 elongase pathway.Additional metabolic engineering and fermentation methods are providedto further enhance DHA productivity in this oleaginous yeast, as well asmethodology to enable production of DHA via the Δ9 elongase/Δ8desaturase pathway (thereby producing DHA-containing oil that is devoidof GLA).

SUMMARY OF THE INVENTION

The present invention relates to recombinant production hosts engineeredto produce docosahexaenoic acid (DHA), methods of making the same andfood feed products containing the microbial oils produced by therecombinant hosts of the invention.

Accordingly, in one embodiment the invention provides recombinantproduction host cell for the production of docosahexaenoic acidcomprising a background Yarrowia sp. comprising a gene pool comprisingthe following genes of the ω-3/ω-6 fatty acid biosynthetic pathway:

a) at least one gene encoding Δ6 desaturase;

b) at least one gene encoding C_(18/20) elongase;

c) at least one gene encoding Δ5 desaturase;

d) at least one gene encoding Δ17 desaturase;

e) at least one gene encoding C_(20/22) elongase; and, f) at least onegene encoding Δ4 desaturase.

In another embodiment the invention provides A recombinant productionhost cell for the production of docosahexaenoic acid comprising abackground Yarrowia sp. comprising a gene pool comprising the followinggenes of the ω-3/ω-6 fatty acid biosynthetic pathway:

a) at least one gene encoding Δ15 desaturase;

b) at least one gene encoding Δ6 desaturase;

c) at least one gene encoding C_(18/20) elongase;

d) at least one gene encoding Δ5 desaturase;

e) at least one gene encoding C_(20/22) elongase; and,

f) at least one gene encoding Δ4 desaturase.

In another embodiment the invention provides a recombinant productionhost cell for the production of docosahexaenoic acid comprising abackground Yarrowia sp. comprising a gene pool comprising the followinggenes of the ω-3/ω-6 fatty acid biosynthetic pathway:

a) at least one gene encoding Δ9 elongase;

b) at least one gene encoding Δ8 desaturase;

c) at least one gene encoding Δ5 desaturase;

d) at least one gene encoding Δ17 desaturase;

e) at least one gene encoding C_(20/22) elongase; and

f) at least one gene encoding Δ4 desaturase.

In an alternate embodiment the invention provides a recombinantproduction host cell for the production of docosahexaenoic acidcomprising a background Yarrowia sp. comprising a gene pool comprisingthe following genes of the ω-3/ω-6 fatty acid biosynthetic pathway:

a) at least one gene encoding Δ15 desaturase;

b) at least one gene encoding Δ9 elongase;

c) at least one gene encoding Δ8 desaturase;

d) at least one gene encoding Δ5 desaturase;

e) at least one gene encoding C_(20/22) elongase; and

f) at least one gene encoding Δ4 desaturase.

In a preferred embodiment the production host of the inventionoptionally comprises at least one gene encoding a Δ12 desaturase. Inanother preferred embodiment the invention provides a recombinantproduction host producing a microbial oil having at least about 5%docosahexaenoic acid as a percent of the total fatty acids.

In another embodiment the invention provides A method for the productionof a microbial oil comprising docosahexaenoic acid comprising:

a) culturing the production host of the invention wherein a microbialoil comprising docosahexaenoic acid is produced; and

b) optionally recovering the microbial oil of step (a).

In another embodiment the invention provides microbial oils made buy themethods and production hosts of the invention. In a preferred embodimentthe microbial oils of the invention contain DHA but are devoid of anyγ-linoleic acid.

In another embodiment the invention provides a food product comprisingan effective amount of a microbial oil produced by the methods of theinvention. Alternatively the invention provides a product selected fromthe group consisting of a medical food, a dietary supplement; an infantformula and a pharmaceutical comprising an effective amount of amicrobial oil produced by the methods of the invention.

Alternatively the invention provides an animal feed comprising aneffective amount of the microbial oil produced by the methods of theinvention.

In another embodiment the invention provides a method for providing ahuman, animal or aquaculture organism diet supplement enriched witheicosapentaenoic acid comprising providing a microbial oil produced bythe methods of the invention containing docosahexaenoic acid in a formconsumable or usable by humans or animals.

Alternatively the invention provides a method for treating a deficiencyin docosahexaenoic acid in animals or humans comprising providing amicrobial oil produced by the methods of the invention containingdocosahexaenoic acid in a form consumable or usable by humans or animalsto treat said deficiency

BIOLOGICAL DEPOSITS

The following biological materials have been deposited with the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110-2209, and bear the following designations, accession numbersand dates of deposit.

Biological Material Accession Number Date of Deposit Plasmid pY89-5 ATCCPTA-6048 June 4^(th), 2004 Yarrowia lipolytica Y2047 ATCC PTA- Yarrowialipolytica Y2096 ATCC PTA- Yarrowia lipolytica Y2201 ATCC PTA- Yarrowialipolytica Y3000 ATCC PTA-

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

FIG. 1 illustrates the ω-3/ω-6 fatty acid biosynthetic pathway.

FIG. 2 is a schematic illustration of the biochemical mechanism forlipid accumulation in oleaginous yeast.

FIG. 3A shows a phylogenetic tree of Δ12 desaturase and Δ15 desaturaseproteins from different filamentous fungi and created using MegalignDNASTAR software. FIG. 3B provides a plasmid map for pY57.YI.AHAS.w497I.

FIG. 4 is a schematic illustration describing the role of variousacyltransferases in lipid accumulation in oleaginous yeast.

FIG. 5 diagrams the development of some Yarrowia lipolytica strains ofthe invention, producing various fatty acids (including DHA) in thetotal lipid fraction.

FIG. 6A provides a plasmid map for pY5-30. FIG. 6B illustrates therelative promoter activities of TEF, GPD, GPM, FBA and FBAIN in Yarrowialipolytica ATCC #76982 strains, as determined by histochemical staining.FIG. 6C illustrates the relative promoter activities of YAT1, TEF, GPATand FBAIN in Y. lipolytica grown in various media as determined byhistochemical staining.

FIG. 7A is a graph comparing the promoter activity of GPD, GPM, FBA andFBAIN in Yarrowia lipolytica ATCC #76982 strains, as determinedfluorometrically. FIG. 7B graphically summarizes the results of RealTime PCR relative quanitation, wherein the GUS mRNA in Yarrowialipolytica ATCC #76982 strains (i.e., expressing GPD::GUS, GPDIN::GUS,FBA::GUS or FBAIN::GUS chimeric genes) was quantified to the mRNA levelof the Y. lipolytica strain expressing pY5-30 (i.e., a chimeric TEF::GUSgene).

FIG. 8 provides plasmid maps for the following: (A) pKUNF12T6E; (B)pDMW271; (C) pZP3L37; (D) pZKUT16; and (E) pKO2UM25E.

FIG. 9 provides plasmid maps for the following: (A) pDMW303; (B) pZUF17;(C) pZUF4; (D) pFOE2S; and (E) pZP2FOEN4.

FIG. 10 provides plasmid maps for the following: (A) pKUNT2; (B)pDMW237; (C) pDMW240; (D) yeast expression vector pY89-5; and (E)pKUNFmKF2.

FIG. 11 shows a chromatogram of the lipid profile of an Euglena graciliscell extract

FIG. 12 shows an alignment of various Euglena gracilis Δ8 desaturasepolypeptide sequences. The method of alignment used corresponds to the“Clustal V method of alignment”.

FIG. 13 provides plasmid maps for the following: (A) pDMW277; (B)pZF5T-PPC; (C) pDMW287F; and (D) pDMW297.

FIG. 14 provides plasmid maps for the following: (A) pZP2C16M899; (B)pDMW314; (C) pDM325; and (D) pZKL5598.

FIG. 15 provides plasmid maps for the following: (A) pY72 [or“pY72.2loxp.Hyg.Fba.F15”]; (B) pY80 [or “pY80.loxp.2F15”]; (C) pY79 [or“pY79.Cre.AHASw497L”; and (D) pY86 [or “pY86.loxp.Ura3.Hyg.F12”].

FIG. 16 provides plasmid maps for the following: (A) pY94 [or“pY94.loxp.D9ED8.Ura3”]; (B) pY91M [or “pY91.Dr.D6M (native)”]; (C)pDMW232; and (D) pY37/F15.

FIG. 17 provides plasmid maps for the following: (A) pKO2UF2PE; (B)pZKUGPI5S; (C) pDMW302T16; and (D) pKO2UM26E.

FIG. 18 provides plasmid maps for the following: (A) pZUF-Mod-1; (B)pMDAGAT1-17; and (C) pMGPAT-17.

FIG. 19 graphically represents the relationship between SEQ ID NOs:138,139, 140, 141, 142, 143, 144, 145, 146, 147 and 148, each of whichrelates to glycerol-3-phosphate o-acyltransferase (GPAT) in Mortierellaalpina.

FIG. 20 graphically represents the relationship between SEQ ID NOs:86,87, 88, 89, 90, 91, 92 and 93, each of which relates to the C16/18 fattyacid elongase enzyme (ELO3) in Mortierella alpina.

FIG. 21 provides plasmid maps for the following: (A) pZUF6S; (B)pZUF6S-E3WT; (C) pZKUGPYE1-N; and (D) pZKUGPYE2.

FIG. 22 provides plasmid maps for the following: (A) pZKUGPYE1; (B)pZUF6FYE1; (C) pZP2I7+Ura; (D) pY20; and (E) pLV13.

The invention can be more fully understood from the following detaileddescription and the accompanying sequence descriptions, which form apart of this application.

The following sequences comply with 37 C.F.R. §1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

SEQ ID NOs:1-153 and 210-221 are ORFs encoding promoters, genes orproteins (or fragments thereof) as identified in Table 1.

TABLE 1 Summary of Gene and Protein SEQ ID Numbers Nucleic acid ProteinDescription SEQ ID NO. SEQ ID NO. Mortierella alpina Δ6 desaturase  1(1374 bp)  2 (457 AA) Synthetic Δ6 desaturase, derived from  3 (1374 bp) 2 (457 AA) Mortierella alpina, codon-optimized for expression inYarrowia lipolytica Mortierella alpina Δ6 desaturase “B”  4 (1521 bp)  5(458 AA) Mortierella alpina Δ5 desaturase  6 (1341 bp)  7 (446 AA)Isochrysis galbana Δ5 desaturase  8 (1329 bp)  9 (442 AA) Synthetic Δ5desaturase derived from  10 (1329 bp)  9 (442 AA) Isochrysis galbana,codon-optimized for expression in Yarrowia lipolytica Homo sapiens Δ5desaturase  11 (1335 bp)  12 (444 AA) Synthetic Δ5 desaturase derivedfrom  13 (1335 bp)  12 (444 AA) Homo sapiens, codon-optimized forexpression in Yarrowia lipolytica Danio rerio Δ5/Δ6 desaturase  14 (1590bp)  15 (444 AA) Drd6/d5(V) (GenBank Accession No. AF309556) Danio rerioΔ5/Δ6 desaturase  16 (1946 bp) — (GenBank Accession No. BC068224) Daniorerio Δ5/Δ6 desaturase mutant  17 (1335 bp)  18 (444 AA) Drd6/d5(M)Saprolegnia diclina Δ17 desaturase  19 (1077 bp)  20 (358 AA) SyntheticΔ17 desaturase gene derived  21 (1077 bp)  20 (358 AA) from Saprolegniadiclina, codon- optimized for expression in Yarrowia lipolyticaMortierella alpina C_(18/20) elongase  22 (957 bp)  23 (318 AA)Synthetic C_(18/20) elongase gene derived  24 (957 bp)  23 (318 AA) fromMortierella alpina, codon- optimized for expression in Yarrowialipolytica Thraustochytrium aureum C_(18/20)  25 (819 bp)  26 (272 AA)elongase Synthetic C_(18/20) elongase gene derived  27 (819 bp)  26 (272AA) from Thraustochytrium aureum, codon- optimized for expression inYarrowia lipolytica Yarrowia lipolytica Δ12 desaturase  28 (1936 bp)  29(419 AA) Mortierella isabellina Δ12 desaturase  30 (1203 bp)  31 (400AA) Fusarium moniliforme Δ12 desaturase  32 (1434 bp)  33 (477 AA)Aspergillus nidulans Δ12 desaturase  34 (1416 bp)  35 (471 AA)Aspergillus flavus Δ12 desaturase —  36 (466 AA) Aspergillus fumigatusΔ12 desaturase —  37 (424 AA) Magnaporthe grisea Δ12 desaturase  38(1656 bp)  39 (551 AA) Neurospora crassa Δ12 desaturase  40 (1446 bp) 41 (481 AA) Fusarium graminearium Δ12  42 (1371 bp)  43 (456 AA)desaturase Mortierella alpina Δ12 desaturase  44 (1403 bp)  45 (400 AA)Saccharomyces kluyveri Δ12 —  46 (416 AA) desaturase Kluyveromyceslactis Δ12 desaturase  47 (1948 bp)  48 (415 AA) Candida albicans Δ12desaturase —  49 (436 AA) Debaryomyces hansenii CBS767 Δ12 —  50 (416AA) desaturase Fusarium moniliforme Δ15 desaturase  51 (1209 bp)  52(402 AA) Aspergillus nidulans Δ15 desaturase  53 (1206 bp)  54 (401 AA)Magnaporthe grisea Δ15 desaturase  55 (1185 bp)  56 (394 AA) Neurosporacrassa Δ15 desaturase  57 (1290 bp)  58 (429 AA) Fusarium gramineariumΔ15  59 (1212 bp)  60 (403 AA) desaturase Mortierella alpina Δ15desaturase  61 (1353 bp)  62 (403 AA) Kluyveromyces lactis Δ15desaturase  63 (1248 bp)  64 (415 AA) Candida albicans Δ15 desaturase — 65 (433 AA) Saccharomyces kluyveri Δ15 —  66 (419 AA) desaturaseDebaryomyces hansenii CBS767 Δ15 —  67 (435 AA) desaturase Aspergillusfumigatus Δ15 desaturase —  68 (396 AA) Isochrysis galbana Δ9 elongase 69 (792 bp)  70 (263 AA) Synthetic Δ9 elongase gene, codon-  71 (792bp)  70 (263 AA) optimized for expression in Yarrowia lipolytica Euglenagracillis Δ8 desaturase gene  72 (1275 bp)  73 (419 AA) (non-functional;GenBank Accession No. AAD45877) Euglena gracillis Δ8 desaturase gene — 74 (422 AA) (non-functional; Wallis et al. [Archives of Biochem.Biophys., 365: 307-316 (1999)]; WO 00/34439) Synthetic Δ8 desaturasegene, codon-  75 (1270 bp) — optimized for expression in Yarrowialipolytica (D8S-1) Synthetic Δ8 desaturase gene, codon-  76 (1269 bp) —optimized for expression in Yarrowia lipolytica (D8S-3) Euglenagracillis Δ8 desaturase gene  77 (1271 bp)  78 (421 AA) (Eg5) Euglenagracillis Δ8 desaturase gene  79 (1271 bp)  80 (421 AA) (Eg12) SyntheticΔ8 desaturase gene, codon-  81 (1272 bp)  82 (422 AA) optimized forexpression in Yarrowia lipolytica (D8SF) Rattus norvegicus C_(16/18)elongase  83 (2628 bp)  84 (267 AA) Synthetic C_(16/18) elongase genederived  85 (804 bp)  84 (267 AA) from Rattus norvegicus, codon-optimized for expression in Yarrowia lipolytica Mortierella alpinaC_(16/18) elongase  86 (828 bp)  87 (275 AA) (ELO3) Mortierella alpinaELO3-partial  88 (607 bp) — cDNA sequence Mortierella alpina ELO3-3′sequence  89 (1042 bp) — obtained by genome walking Mortierella alpinaELO3-5′ sequence  90 (2223 bp) — obtained by genome walking Mortierellaalpina ELO3-cDNA  91 (3557 bp) — contig Mortierella alpina ELO3-intron 92 (542 bp) — Mortierella alpina ELO3-genomic  93 (4099 bp) — contigYarrowia lipolytica C_(16/18) elongase  94 (915 bp)  95 (304 AA) geneCandida albicans probable fatty acid —  96 (353 AA) elongase (GenBankAccession No. EAL04510) Yarrowia lipolytica C_(14/16) elongase  97 (978bp)  98 (325 AA) gene Neurospora crassa FEN1 gene —  99 (337 AA)(GenBank Accession No. CAD70918) Ostreococcus tauri C_(20/22) elongase100 (903 bp) 101 (300 AA) Synthetic C_(20/22) elongase gene derived 102(903 bp) 103 (300 AA) from Ostreococcus tauri, codon- optimized forexpression in Yarrowia lipolytica Thraustochytrium aureum Δ4 104 (1548bp) 105 (515 AA) desaturase Synthetic Δ4 desaturase gene derived 106(1545 bp) 107 (514 AA) from Thraustochytrium aureum, codon- optimizedfor expression in Yarrowia lipolytica Mortierella alpinalysophosphatidic 108 (945 bp) 109 (314 AA) acid acyltransferase (LPAAT1)Mortierella alpina lysophosphatidic 110 (927 bp) 111 (308 AA) acidacyltransferase (LPAAT2) Yarrowia lipolytica lysophosphatidic 112 (1549bp) 113 (282 AA) acid acyltransferase (LPAAT1) Yarrowia lipolyticalysophosphatidic 114 (1495 bp) — acid acyltransferase (LPAAT2)- genomicfragment comprising gene Yarrowia lipolytica lysophosphatidic 115 (672bp) 116 (223 AA) acid acyltransferase (LPAAT2) Yarrowia lipolytica 117(2326 bp) 118 (648 AA) phospholipid:diacylglycerol acyltransferase(PDAT) Yarrowia lipolytica acyl-CoA:sterol- 119 (1632 bp) 120 (543 AA)acyltransferase (ARE2) Caenorhabditis elegans acyl-CoA: 1- — 121 (282AA) acyl lysophosphatidylcholine acyltransferase (LPCAT) Yarrowialipolytica diacylglycerol 122 (1578 bp) 123 (526 AA) acyltransferase(DGAT1) Mortierella alpina diacylglycerol 124 (1578 bp) 125 (525 AA)acyltransferase (DGAT1) Neurospora crassa diacylglycerol — 126 (533 AA)acyltransferase (DGAT1) Gibberella zeae PH-1 diacylglycerol — 127 (499AA) acyltransferase (DGAT1) Magnaporthe grisea diacylglycerol — 128 (503AA) acyltransferase (DGAT1) Aspergillus nidulans diacylglycerol — 129(458 AA) acyltransferase (DGAT1) Yarrowia lipolytica diacylglycerol 130(2119 bp) 131 (514 AA) acyltransferase (DGAT2) 132 (1380 bp) 133 (459AA) 134 (1068 bp) 135 (355 AA) Mortierella alpina diacylglycerol 136(996 bp) 137 (331 AA) acyltransferase (DGAT2) Mortierella alpinaglycerol-3-phosphate 138 (2151 bp) 139 (716 AA) acyltransferase (GPAT)M. alpina GPAT-partial cDNA 140 (1212 bp) — sequence M. alpinaGPAT-genomic fragment 141 (3935 bp) — comprising −1050 bp to + 2886 bpregion M. alpina GPAT-3′ cDNA sequence 142 (965 bp) — obtained by genomewalking M. alpina GPAT-5′ sequence 143 (1908 bp) — obtained by genomewalking M. alpina GPAT-internal sequence 144 (966 bp) — obtained bygenome walking M. alpina GPAT-intron #1 145 (275 bp) — M. alpinaGPAT-intron #2 146 (255 bp) — M. alpina GPAT-intron #3 147 (83 bp) — M.alpina GPAT-intron #4 148 (99 bp) — Yarrowia lipolytica diacylglycerol149 (2133 bp) — cholinephosphotransferase (CPT1) - genomic fragmentcomprising gene Yarrowia lipolytica diacylglycerol 150 (1185 bp) 151(394 AA) cholinephosphotransferase (CPT1) Saccharomyces cerevisiaeinositol 152 (1434 bp) 153 (477 AA) phosphosphingolipid-specificphospholipase C (ISC1) Yarrowia lipolytica glyceraldehyde-3- 210 —phosphate dehydrogenase promoter (971 bp) (GPD) Yarrowia lipolyticaglyceraldehyde-3- 211 — phosphate dehydrogenase + intron (1174 bp)promoter (GPDIN) Yarrowia lipolytica phosphoglycerate 212 — mutasepromoter (GPM) (878 bp) Yarrowia lipolytica fructose- 213 — bisphosphatealdolase promoter (FBA) (1001 bp) Yarrowia lipolytica fructose- 214 —bisphosphate aldolase + intron (973 bp) promoter (FBAIN) Yarrowialipolytica fructose- 215 — bisphosphate aldolase + modified (924 bp)intron promoter (FBAINm) Yarrowia lipolytica glycerol-3- 216 — phosphateacyltransferase promoter (1130 bp) (GPAT) Yarrowia lipolytica ammonium217 — transporter promoter (YAT1) (778 bp) Yarrowia lipolyticatranslation 218 — elongation factor EF1-α promoter (436 bp) (TEF)Yarrowia lipolytica chimeric 219 — GPM::FBA intron promoter (1020 bp)(GPM::FBAIN) Yarrowia lipolytica chimeric 220 — GPM::GPD intron promoter(1052 bp) (GPM::GPDIN) Yarrowia lipolytica export protein 221 — promoter(EXP1) (1000 bp)

SEQ ID NOs: 154 and 156-209 are plasmids as identified in Table 2.

TABLE 2 Summary of Plasmid SEQ ID Numbers Plasmid Corresponding FigureSEQ ID NO pY5-30  6A 154 (8,953 bp) pKUNF12T6E  8A 156 (12,649 bp)pDMW271  8B 157 (13,034 bp) pZP3L37  8C 158 (12,690 bp) pZKUT16  8D 159(5,833 bp) pKO2UM25E  8E 160 (12,663 bp) pDMW303  9A 161 (15,996 bp)pZUF17  9B 162 (8,165 bp) pZUF4 — 163 (8,633 bp) pZUF4S  9C 164 (8,633bp) pZP2FOEN4  9E 165 (10,660 bp) pKUNT2 10A 166 (6,457 bp) pDMW237 10B167 (7,879 bp) pY54PC — 168 (8,502 bp) pKUNFmkF2 10E 169 (7,145 bp)pZF5T-PPC 13B 170 (5,553 bp) pDMW297 13D 171 (10,448 bp) pZP2C16M899 14A172 (15,543 bp) pDMW314 14B 173 (13,295 bp) pDMW325 14C 174 (15,559 bp)pZKSL5598 14D 175 (16,325 bp) pY72 15A 176 (10,189 bp) pY80 15B 177(12,558 bp) pY79 15C 178 (8,982 bp) pY86 15D 179 (10,424 bp) pY94 16A180 (10,485 bp) pY91M 16B 181 (8,423 bp) pDMW232 16C 182 (10,945 bp)pY37/F15 16D 183 (8,194 bp) pKO2UF2PE 17A 184 (10,838 bp) pZKUGPI5S 17B185 (6,912 bp) pDMW302T16 17C 186 (14,864 bp) pZKUGPE1S — 187 (6,540 bp)pKO2UM26E 17D 188 (13,321 bp) pZKUM — 189 (4,313 bp) pMLPAT-17 — 190(8,015 bp) pMLPAT-Int — 191 (8,411 bp) pZUF-MOD-1 18A 192 (7,323 bp)pMDGAT1-17 18B 193 (8,666 bp) pMDGAT2-17 — 194 (8,084 bp) pMGPAT-17 18C195 (9,239 bp) pZF5T-PPC-E3 — 196 (5,031 bp) pZUF6S 21A 197 (8,462 bp)pZUF6S-E3WT 21B 198 (11,046 bp) pZKUGPYE1-N 21C 199 (6,561 bp) pZKUGPYE221D 200 (6,498 bp) pZUF6TYE2 — 201 (10,195 bp) pZKUGPYE1 22A 202 (6,561bp) pZUF6FYE1 22B 203 (10,809 bp) pYCPT1-17 — 204 (8,273 bp) pZP2I7 +Ura 22C 205 (7,822 bp) pYCPT1-ZP2I7 — 206 (7,930 bp) pTEF::ISC1 — 207(8,179 bp) pY20 22D 208 (8,196 bp) pLV13 22E 209 (5,105 bp)

SEQ ID NO:155 corresponds to the codon-optimized translation initiationsite for genes optimally expressed in Yarrowia sp.

SEQ ID NO:222 corresponds to a His Box 1 motif found in fungal Δ15 andΔ12 desaturases.

SEQ ID NO:223 corresponds to a motif that is indicative of a fungalprotein having Δ15 desaturase activity, while SEQ ID NO:224 correspondsto a motif that is indicative of a fungal protein having Δ12 desaturaseactivity.

SEQ ID NOs:225-238 correspond to primers YL211, YL212, YL376, YL377,YL203, YL204, GPAT-5-1, GPAT-5-2, ODMW314, YL341, ODMW320, ODMW341,27203-F and 27203-R, respectively, used to amplify Yarrowia lipolyticapromoter regions.

SEQ ID NOs:239-242 are the oligonucleotides YL-URA-16F, YL-URA-78R,GUS-767F and GUS-891 R, respectively, used for Real Time analysis.

SEQ ID NO:243 is a mutant AHAS gene comprising a W497L mutation.

SEQ ID NOs:244-249 correspond to primers 410, 411, 412, 413, 414 and415, respectively, used for synthesis of a mutant Yarrowia lipolyticaAHAS gene, comprising a W497L mutation.

SEQ ID NOs:250-281 correspond to 16 pairs of oligonucleotides whichtogether comprise the entire codon-optimized coding region of theThraustochytrium aureum Δ4 desaturase (i.e., D4-1A, D4-1B, D4-2A, D4-2B,D4-3A, D4-3B, D4-4A, D4-4B, D4-5A, D4-5B, D4-6A, D4-6B, D4-7A, D4-7B,D4-8A, D4-8B, D4-9A, D4-9B, D4-10A, D4-10B, D4-11A, D4-11B, D4-12A,D4-12B, D4-13A, D4-13B, D4-14A, D4-14B, D4-15A, D4-15B, D4-16A andD4-16B).

SEQ ID NOs:282-289 correspond to primers D4-1F, D4-4R, D4-5F, D4-8R,D4-9F, D4-12R, D4-13 and D4-16R, respectively, used for PCRamplification during synthesis of the codon-optimized Δ4 desaturasegene.

SEQ ID NOs:290 and 291 correspond to primers YL251 and YL252,respectively, used during synthesis of the codon-optimized Δ4 desaturasegene.

SEQ ID NOs:292-307 correspond to 8 pairs of oligonucleotides whichtogether comprise the entire codon-optimized coding region of the I.galbana Δ9 elongase (i.e., IL3-1A, IL3-1B, IL3-2A, IL3-2B, IL3-3A,IL3-3B, IL3-4A, IL3-4B, IL3-5A, IL3-5B, IL3-6A, IL3-6B, IL3-7A, IL3-7B,IL3-8A and IL3-8B, respectively).

SEQ ID NOs:308-311 correspond to primers IL3-1 F, IL3-4R, IL3-5F andIL3-8R, respectively, used for PCR amplification during synthesis of thecodon-optimized Δ9 elongase gene.

SEQ ID NO:312 is the 417 bp NcoI/PstI fragment described in pT9(1-4);and SEQ ID NO:313 is the 377 bp PstI/Not1 fragment described inpT9(5-8).

SEQ ID NOs:314-339 correspond to 13 pairs of oligonucleotides whichtogether comprise the entire codon-optimized coding region of the E.gracilis Δ8 desaturase (i.e., D8-1A, D8-1B, D8-2A, D8-2B, D8-3A, D8-3B,D8-4A, D8-4B, D8-5A, D8-5B, D8-6A, D8-6B, D8-7A, D8-7B, D8-8A, D8-8B,D8-9A, D8-9B, D8-10A, D8-10B, D8-11A, D8-11B, D8-12A, D8-12B, D8-13A andD8-13B, respectively).

SEQ ID NOs:340-347 correspond to primers D8-1F, D8-3R, D8-4F, D8-6R,D8-7F, D8-9R, D8-10F and D8-13R, respectively, used for PCRamplification during synthesis of the codon-optimized Δ8 desaturasegene.

SEQ ID NO:348 is the 309 bp Nco/BgIII fragment described in pT8(1-3);SEQ ID NO:349 is the 321 bp BgIII/XhoI fragment described in pT8(4-6);SEQ ID NO:350 is the 264 bp XhoI/SacI fragment described in pT8(7-9);and SEQ ID NO:351 is the 369 bp Sac1/Not1 fragment described inpT8(10-13).

SEQ ID NOs:352 and 353 correspond to primers ODMW390 and ODMW391,respectively, used during synthesis of D8S-2 in pDMW255.

SEQ ID NOs:354 and 355 are the chimeric D8S-1::XPR and D8S-2::XPR genesdescribed in Example 9.

SEQ ID NOs:356 and 357 correspond to primers ODMW392 and ODMW393, usedduring synthesis of D8S-3.

SEQ ID NOs:358 and 359 correspond to primers Eg5-1 and Eg3-3,respectively, used for amplification of the Δ8 desaturase from Euglenagracilis.

SEQ ID NOs:360-363 correspond to primers T7, M13-28Rev, Eg3-2 and Eg5-2,respectively, used for sequencing a Δ8 desaturase clone.

SEQ ID NO:364 corresponds to primer ODMW404, used for amplification ofD8S-3.

SEQ ID NO:365 is a 1272 bp chimeric gene comprising D8S-3.

SEQ ID NOs:366 and 367 correspond to primers YL521 and YL522,respectively, used to create new restriction enzyme sites in a clonedD8S-3 gene.

SEQ ID NOs:368-381 correspond to primers YL525, YL526, YL527, YL528,YL529, YL530, YL531, YL532, YL533, YL534, YL535, YL536, YL537 and YL538,respectively, used in site directed mutagenesis reactions to produceD8SF.

SEQ ID NO:382 corresponds to a LoxP recombination site that isrecognized by the Cre recombinase enzyme.

SEQ ID NOs:383 and 384 correspond to primers 436 and 437, respectively,used to amplify a GPD::Fm1::XPR2 during synthesis of plasmid pY80.

SEQ ID NOs:385-388 correspond to primers 475, 477, 478 and 476,respectively, used to clone a bifunctional Δ5/Δ6 desaturase.

SEQ ID NOs:389 and 390 correspond to primers 505 and 506, respectively,used to created plasmid pY91V from plasmid pY91M by site-specificmutagenesis.

SEQ ID NOs:391-393 correspond to BD-Clontech Creator Smart® cDNA librarykit primers SMART IV oligonucleotide, CDSIII/3′ PCR primer and 5′-PCRprimer, respectively.

SEQ ID NO:394 corresponds to the M13 forward primer used for M. alpinacDNA library sequencing.

SEQ ID NOs:395-398 and 400-401 correspond to primers MLPAT-F, MLPAT-R,LPAT-Re-5-1, LPAT-Re-5-2, LPAT-Re-3-1 and LPAT-Re-3-2, respectively,used for cloning of the M. alpina LPAAT2 ORF.

SEQ ID NOs:399 and 402 correspond to a 5′ (1129 bp) and 3′ (938 bp)region of the Y. lipolytica LPAAT1 ORF, respectively.

SEQ ID NOs:403 and 404 correspond to primers pzuf-mod1 and pzuf-mod2,respectively, used for creating “control” plasmid pZUF-MOD-1.

SEQ ID NOs:405 and 406 correspond to primers MACAT-F1 and MACAT-R,respectively, used for cloning of the M. alpina DGAT1 ORF.

SEQ ID NOs:407 and 408 correspond to primers MDGAT-F and MDGAT-R1,respectively, used for cloning of the M. alpina DGAT2 ORF.

SEQ ID NOs:409 and 410 correspond to primers MGPAT-N1 and MGPAT-NR5,respectively, used for degenerate PCR to amplify the M. alpina GPAT.

SEQ ID NOs:411-413 correspond to primers MGPAT-5N1, MGPAT-5N2 andMGPAT-5N3, respectively, used for amplification of the 3′-end of the M.alpina GPAT.

SEQ ID NOs:414 and 415 correspond to the Genome Walker adaptor fromClonTech's Universal GenomeWalker™ Kit, used for genome-walking.

SEQ ID NOs:416-419 correspond to the PCR primers used in genome-walking:MGPAT-5-1A, Adaptor-1 (AP1), MGPAT-3N1 and Nested Adaptor Primer 2(AP2), respectively.

SEQ ID NOs:420 and 421 correspond to primers mgpat-cdna-5 andmgpat-cdna-R, respectively, used for amplifying the M. alpina GPAT.

SEQ ID NOs:422 and 423 correspond to primers MA Elong 3′1 and MA elong3′2, respectively, used for genome-walking to isolate the 3′-end regionof the M. alpina ELO3.

SEQ ID NOs:424 and 425 correspond to primers MA Elong 5′1 and MA Elong5′2, respectively, used for genome-walking to isolate the 5′-end regionof the M. alpina ELO3.

SEQ ID NOs:426 and 427 correspond to primers MA ELONG 5′ NcoI 3 and MAELONG 3′ NotI 1, respectively, used for amplifying the complete ELO3from M. alpina cDNA.

SEQ ID NOs:428 and 429 correspond to primers YL597 and YL598,respectively, used for amplifying the coding region of Y. lipolyticaYE2.

SEQ ID NOs:430 and 431 correspond to primers YL325 and YL326,respectively, used to amplify a NotI/PacI fragment containing the Aco 3′terminator.

SEQ ID NOs:432-435 correspond to primers YL567, YL568, YL569 and YL570,respectively, used for amplifying the coding region of Y. lipolyticaYE1.

SEQ ID NOs:436 and 437 correspond to primers YL571 and YL572,respectively, used for site-directed mutagenesis during cloning of Y.lipolytica YE1.

SEQ ID NOs:438 and 439 correspond to primers CPT1-5′-NcoI andCPT1-3′-NotI, respectively, used for cloning of the Y. lipolytica CPT1ORF.

SEQ ID NOs:440 and 441 correspond to primers Isc1F and Isc1R,respectively, used for cloning of the S. cerevisiae ISC1 ORF.

SEQ ID NOs:442 and 443 correspond to primers PcI1F and PcI1R,respectively, used for cloning of the S. cerevisiae PCL1 ORF.

SEQ ID NOs:444-447 correspond to primers P95, P96, P97 and P98,respectively, used for targeted disruption of the Y. lipolytica DGAT2gene.

SEQ ID NOs:448-450 correspond to primers P115, P116 and P112,respectively, used to screen for targeted integration of the disruptedY. lipolytica DGAT2 gene.

SEQ ID NOs:451-454 correspond to primers P39, P41, P40 and P42,respectively, used for targeted disruption of the Y. lipolytica PDATgene.

SEQ ID NOs:455-458 correspond to primers P51, P52, P37 and P38,respectively, used to screen for targeted integration of the disruptedY. lipolytica PDAT gene.

SEQ ID NOs:459 and 460 are the degenerate primers identified as P201 andP203, respectively, used for the isolation of the Y. lipolytica DGAT1.

SEQ ID NOs:461-465 correspond to primers P214, P215, P216, P217 andP219, respectively, used for the creation of a targeting cassette fortargeted disruption of the putative DGAT1 gene in Y. lipolytica.

SEQ ID NOs:466 and 467 correspond to primers P226 and P227,respectively, used to screen for targeted integration of the disruptedY. lipolytica DGAT1 gene.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, and publications cited herein areincorporated by reference in their entirety including:

U.S. patent application Ser. No. 10/840,478 (filed May 6, 2004),

U.S. patent application Ser. No. 10/840,579 (filed May 6, 2004),

U.S. patent application Ser. No. 10/840,325 (filed May 6, 2004)

U.S. patent application Ser. No. 10/869,630 (filed Jun. 16, 2004),

U.S. patent application Ser. No. 10/882,760 (filed Jul. 1, 2004),

U.S. patent application Ser. No. 10/985,109 (filed Nov. 10, 2004),

U.S. patent application Ser. No. 10/987,548 (filed Nov. 12, 2004)

U.S. Patent Application No. 60/624,812 (filed Nov. 4, 2004),

U.S. patent application Ser. No. 11/024,545 and No. 11/024,544 (filedDec. 29, 2004),

U.S. Patent Application No. 60/689,031 (filed Jun. 9, 2005),

U.S. patent application Ser. No. 11/183,664 (filed Jul. 18, 2005),

U.S. patent application Ser. No. 11/185,301 (filed Jul. 20, 2005),

U.S. patent application Ser. No. 11/190,750 (filed Jul. 27, 2005),

U.S. patent application Ser. No. 11/225,354 (filed Sep. 13, 2005),

U.S. patent application Ser. No. 10/253,882 (filed Oct. 19, 2005)

U.S. patent application Ser. No. 11/254,173 (filed Oct. 19, 2005)

U.S. patent application Ser. No. 10/253,882 (filed Oct. 19, 2005)

U.S. patent application Ser. No. 11/254,173 (filed Oct. 19, 2005).

In accordance with the subject invention, Applicants provide productionhost strains of Yarrowia lipolytica that are capable of producinggreater than 5% docosahexaenoic acid (DHA, 22:6, ω-3). Accumulation ofthis particular polyunsaturated fatty acid (PUFA) is accomplished byintroduction of a functional ω-3/ω-6 fatty acid biosynthetic pathwaycomprising proteins that encode Δ6 desaturase, C_(18/20) elongase, Δ5desaturase, Δ17 desaturase, C_(20/22) elongase and Δ4 desaturaseactivities into the oleaginous yeast host for high-level recombinantexpression. Thus, this disclosure demonstrates that Yarrowia lipolyticacan be engineered to enable commercial production of DHA and derivativesthereof. Methods of production are also claimed.

The subject invention finds many applications. PUFAs, or derivativesthereof, made by the methodology disclosed herein can be used as dietarysubstitutes, or supplements, particularly infant formulas, for patientsundergoing intravenous feeding or for preventing or treatingmalnutrition. Alternatively, the purified PUFAs (or derivatives thereof)may be incorporated into cooking oils, fats or margarines formulated sothat in normal use the recipient would receive the desired amount fordietary supplementation. The PUFAs may also be incorporated into infantformulas, nutritional supplements or other food products and may finduse as anti-inflammatory or cholesterol lowering agents. Optionally, thecompositions may be used for pharmaceutical use (human or veterinary).In this case, the PUFAs are generally administered orally but can beadministered by any route by which they may be successfully absorbed,e.g., parenterally (e.g., subcutaneously, intramuscularly orintravenously), rectally, vaginally or topically (e.g., as a skinointment or lotion).

Supplementation of humans or animals with PUFAs produced by recombinantmeans can result in increased levels of the added PUFAs, as well astheir metabolic progeny. For example, treatment with DHA can result notonly in increased levels of DHA, but also downstream products of DHAsuch as eicosanoids (i.e., prostaglandins, leukotrienes, thromboxanes).Complex regulatory mechanisms can make it desirable to combine variousPUFAs, or add different conjugates of PUFAs, in order to prevent,control or overcome such mechanisms to achieve the desired levels ofspecific PUFAs in an individual.

In alternate embodiments, PUFAs, or derivatives thereof, made by themethodology disclosed herein can be utilized in the synthesis ofaquaculture feeds (i.e., dry feeds, semi-moist and wet feeds) sincethese formulations generally require at least 1-2% of the nutrientcomposition to be ω-3 and/or ω-6 PUFAs.

Definitions

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions are provided.

“Open reading frame” is abbreviated ORF.

“Polymerase chain reaction” is abbreviated PCR.

“American Type Culture Collection” is abbreviated ATCC.

“Polyunsaturated fatty acid(s)” is abbreviated PUFA(s).

“Diacylglycerol acyltransferase” is abbreviated DAG AT or DGAT.

“Phospholipid:diacylglycerol acyltransferase” is abbreviated PDAT.

“Glycerol-3-phosphate acyltransferase” is abbreviated GPAT.

“Lysophosphatidic acid acyltransferase” is abbreviated LPAAT.

“Acyl-CoA:1-acyl lysophosphatidylcholine acyltransferase” is abbreviated“LPCAT”.

“Acyl-CoA:sterol-acyltransferase” is abbreviated ARE2.

“Diacylglycerol” is abbreviated DAG.

“Triacylglycerols” are abbreviated TAGs.

“Co-enzyme A” is abbreviated CoA.

“Phosphatidyl-choline” is abbreviated PC.

The term “Fusarium moniliforme” is synonymous with “Fusariumverticillioides”.

The term “food product” refers to any food generally suitable for humanconsumption. Typical food products include but are not limited to meatproducts, cereal products, baked foods, snack foods, dairy products andthe like.

The term “functional food” refers to those foods that encompasspotentially healthful products including any modified food or ingredientthat may provide a health benefit beyond the traditional nutrients itcontains. Functional foods can include foods like cereals, breads andbeverages which are fortified with vitamins, herbs and nutraceuticals.Functional foods contain a substance that provides health benefitsbeyond its nutritional value, wherein the substance either is naturallypresent in the food or is deliberately added.

As used herein the term “medical food” refers to a food which isformulated to be consumed or administered enterally under thesupervision of a physician and which is intended for the specificdietary management of a disease or condition for which distinctivenutritional requirements, based on recognized scientific principles, areestablished by medical evaluation [see section 5(b) of the Orphan DrugAct (21 U.S.C. 360ee(b)(3))]. A food is a “medical food” only if: (i) Itis a specially formulated and processed product (as opposed to anaturally occurring foodstuff used in its natural state) for the partialor exclusive feeding of a patient by means of oral intake or enteralfeeding by tube; (ii) It is intended for the dietary management of apatient who, because of therapeutic or chronic medical needs, haslimited or impaired capacity to ingest, digest, absorb, or metabolizeordinary foodstuffs or certain nutrients, or who has other specialmedically determined nutrient requirements, the dietary management ofwhich cannot be achieved by the modification of the normal diet alone;(iii) It provides nutritional support specifically modified for themanagement of the unique nutrient needs that result from the specificdisease or condition, as determined by medical evaluation; (iv) It isintended to be used under medical supervision; and (v) It is intendedonly for a patient receiving active and ongoing medical supervisionwherein the patient requires medical care on a recurring basis for,among other things, instructions on the use of the medical food. Thus,unlike dietary supplements or conventional foods, a medical food that isintended for the specific dietary management of a disease or conditionfor which distinctive nutritional requirements have been established,may bear scientifically valid claims relating to providing distinctivenutritional support for a specific disease or condition. Medical foodsare distinguished from the broader category of foods for special dietaryuse (e.g., hypoallergenic foods) and from foods that make health claims(e.g., dietary supplements) by the requirement that medical foods beused under medical supervision.

The term “medical nutritional” is a medical food as defined hereintypically refers to a fortified beverage that is specifically designedfor special dietary needs. The medical nutritional generally comprises adietary composition focused at a specific medical or dietary condition.Examples of commercial medical nuturitionals include, but are notlimited to Ensure® and Boost®.

The term “pharmaceutical” as used herein means a compound or substancewhich if sold in the United States would be controlled by Section 505 or505 of the Federal Food, Drug and Cosmetic Act.

The term “infant formula” means a food which is designed exclusively forconsumption by the human infant by reason of its simulation of humanbreast milk. Typical commercial examples of infant formula include burare not limited to Similac®, and Isomil®.

The term “dietary supplement” refers to a product that: (i) is intendedto supplement the diet and thus is not represented for use as aconventional food or as a sole item of a meal or the diet; (ii) containsone or more dietary ingredients (including, e.g., vitamins, minerals,herbs or other botanicals, amino acids, enzymes and glandulars) or theirconstituents; (iii) is intended to be taken by mouth as a pill, capsule,tablet, or liquid; and (iv) is labeled as being a dietary supplement.

A “food analog” is a food-like product manufactured to resemble its foodcounterpart, whether meat, cheese, milk or the like, and is intended tohave the appearance, taste, and texture of its counterpart. Thus, theterm “food” as used herein also encompasses food analogs.

The terms “aquaculture feed” and “aquafeed” refer to manufactured orartificial diets (formulated feeds) to supplement or to replace naturalfeeds in the aquaculture industry. Thus, an aquafeed refers toartificially compounded feeds that are useful for farmed finfish andcrustaceans (i.e., both lower-value staple food fish species [e.g.,freshwater finfish such as carp, tilapia and catfish] and higher-valuecash crop species for luxury or niche markets [e.g., mainly marine anddiadromous species such as shrimp, salmon, trout, yellowtail, seabass,seabream and grouper]). These formulate feeds are composed of severalingredients in various proportions complementing each other to form anutritionally complete diet for the aquacultured species.

The term “animal feed” refers to feeds intended exclusively forconsumption by animals, including domestic animals (pets, farm animalsetc..) or for animals raised for the production of food e.g. fishfarming.

The term “feed nutrient” means nutrients such as proteins, lipids,carbohydrates, vitamins, minerals, and nucleic acids that may be derivedfrom the yeast biomass comprising the recombinant production hosts ofthe invention.

As used herein the term “biomass” refers specifically to spent or usedyeast cellular material from the fermentation of a recombinantproduction host production EPA in commercially significant amounts. Theterm “fatty acids” refers to long chain aliphatic acids (alkanoic acids)of varying chain lengths, from about C₁₂ to C₂₂ (although both longerand shorter chain-length acids are known). The predominant chain lengthsare between C₁₆ and C₂₂. The structure of a fatty acid is represented bya simple notation system of “X:Y”, where X is the total number of carbon(C) atoms in the particular fatty acid and Y is the number of doublebonds. Additional details concerning the differentiation between“saturated fatty acids” versus “unsaturated fatty acids”,“monounsaturated fatty acids” versus “polyunsaturated fatty acids” (or“PUFAs”), and “omega-6 fatty acids” (ω-6 or ω-6) versus “omega-3 fattyacids” (ω-3 or ω-3) are provided in WO2004/101757.

Nomenclature used to describe PUFAs in the present disclosure is shownbelow in Table 3. In the column titled “Shorthand Notation”, theomega-reference system is used to indicate the number of carbons, thenumber of double bonds and the position of the double bond closest tothe omega carbon, counting from the omega carbon (which is numbered 1for this purpose). The remainder of the Table summarizes the commonnames of ω-3 and ω-6 fatty acids and their precursors, the abbreviationsthat will be used throughout the specification and each compounds'chemical name.

TABLE 3 Nomenclature of Polyunsaturated Fatty Acids And PrecursorsShorthand Common Name Abbreviation Chemical Name Notation Myristic —tetradecanoic 14:0 Palmitate Palmitate hexadecanoic 16:0 Palmitoleic —9-hexadecenoic 16:1 Stearic — octadecanoic 18:0 Oleic —cis-9-octadecenoic 18:1 Linoleic LA cis-9,12-octadecadienoic 18:2 ω-6γ-Linoleic GLA Cis-6,9,12- 18:3 ω-6 octadecatrienoic Eicosadienoic EDAcis-11,14-eicosadienoic 20:2 ω-6 Dihomo-γ- DGLA cis-8,11,14- 20:3 ω-6Linoleic eicosatrienoic Arachidonic ARA cis-5,8,11,14- 20:4 ω-6eicosatetraenoic α-Linolenic ALA cis-9,12,15- 18:3 ω-3 octadecatrienoicStearidonic STA cis-6,9,12,15- 18:4 ω-3 octadecatetraenoicEicosatrienoic ETrA cis-11,14,17- 20:3 ω-3 eicosatrienoic Eicosa- ETAcis-8,11,14,17- 20:4 ω-3 tetraenoic eicosatetraenoic Eicosa- EPAcis-5,8,11,14,17- 20:5 ω-3 pentaenoic eicosapentaenoic Docosa- DPAcis-7,10,13,16,19- 22:5 ω-3 pentaenoic docosapentaenoic Docosa- DHAcis-4,7,10,13,16,19- 22:6 ω-3 hexaenoic docosahexaenoic

The term “high-level DHA production” refers to production of at leastabout 5% DHA in the total lipids of the microbial host, preferably atleast about 10% DHA in the total lipids, more preferably at least about15% DHA in the total lipids, more preferably at least about 20% DHA inthe total lipids and most preferably at least about 25-30% DHA in thetotal lipids. The structural form of the DHA is not limiting; thus, forexample, the DHA may exist in the total lipids as free fatty acids or inesterified forms such as acylglycerols, phospholipids, sulfolipids orglycolipids.

The term “devoid of any GLA” refers to lack of any detectable GLA in thetotal lipids of the microbial host, when measured by GC analysis usingequipment having a detectable level down to about 0.1%.

The term “essential fatty acid” refers to a particular PUFA that anorganism must ingest in order to survive, being unable to synthesize theparticular essential fatty acid de novo. For example, mammals can notsynthesize the essential fatty acids LA (18:2, ω-6) and ALA (18:3, ω-3).Other essential fatty acids include GLA (ω-6), DGLA (ω-6), ARA (ω-6),EPA (ω-3) and DHA (ω-3).

“Microbial oils” or “single cell oils” are those oils naturally producedby microorganisms (e.g., algae, oleaginous yeasts and filamentous fungi)during their lifespan. The term “oil” refers to a lipid substance thatis liquid at 25° C. and usually polyunsaturated. In contrast, the term“fat” refers to a lipid substance that is solid at 25° C. and usuallysaturated.

“Lipid bodies” refer to lipid droplets that usually are bounded byspecific proteins and a monolayer of phospholipid. These organelles aresites where most organisms transport/store neutral lipids. Lipid bodiesare thought to arise from microdomains of the endoplasmic reticulum thatcontain TAG-biosynthesis enzymes; and, their synthesis and size appearto be controlled by specific protein components.

“Neutral lipids” refer to those lipids commonly found in cells in lipidbodies as storage fats and oils and are so called because at cellularpH, the lipids bear no charged groups. Generally, they are completelynon-polar with no affinity for water. Neutral lipids generally refer tomono-, di-, and/or triesters of glycerol with fatty acids, also calledmonoacylglycerol, diacylglycerol or TAG, respectively (or collectively,acylglycerols). A hydolysis reaction must occur to release free fattyacids from acylglycerols.

The terms “triacylglycerol”, “oil” and “TAGs” refer to neutral lipidscomposed of three fatty acyl residues esterified to a glycerol molecule(and such terms will be used interchangeably throughout the presentdisclosure herein). Such oils can contain long chain PUFAs, as well asshorter saturated and unsaturated fatty acids and longer chain saturatedfatty acids. Thus, “oil biosynthesis” generically refers to thesynthesis of TAGs in the cell.

The term “acyltransferase” refers to an enzyme responsible fortransferring a group other than an amino-acyl group (EC 2.3.1.-).

The term “DAG AT” refers to a diacylglycerol acyltransferase (also knownas an acyl-CoA-diacylglycerol acyltransferase or a diacylglycerolO-acyltransferase) (EC 2.3.1.20). This enzyme is responsible for theconversion of acyl-CoA and 1,2-diacylglycerol to TAG and CoA (therebyinvolved in the terminal step of TAG biosynthesis). Two families of DAGAT enzymes exist: DGAT1 and DGAT2. The former family shares homologywith the acyl-CoA:cholesterol acyltransferase (ACAT) gene family, whilethe latter family is unrelated (Lardizabal et al., J. Biol. Chem.276(42):38862-38869 (2001)).

The term “PDAT” refers to a phospholipid:diacylglycerol acyltransferaseenzyme (EC 2.3.1.158). This enzyme is responsible for the transfer of anacyl group from the sn-2 position of a phospholipid to the sn-3 positionof 1,2-diacylglycerol, thus resulting in lysophospholipid and TAG(thereby involved in the terminal step of TAG biosynthesis). This enzymediffers from DGAT (EC 2.3.1.20) by synthesizing TAG via anacyl-CoA-independent mechanism.

The term “ARE2” refers to an acyl-CoA:sterol-acyltransferase enzyme (EC2.3.1.26; also known as a sterol-ester synthase 2 enzyme), catalyzingthe following reaction: acyl-CoA+sterol=CoA+sterol ester.

The term “GPAT” refers to a glycerol-3-phosphate O-acyltransferaseenzyme (E.C. 2.3.1.15) encoded by the gpat gene and which convertsacyl-CoA and sn-glycerol 3-phosphate to CoA and 1-acyl-sn-glycerol3-phosphate (the first step of phospholipid biosynthesis).

The term “LPAAT” refers to a lysophosphatidic acid-acyltransferaseenzyme (EC 2.3.1.51). This enzyme is responsible for the transfer of anacyl-CoA group onto 1-acyl-sn-glycerol 3-phosphate (i.e.,lysophosphatidic acid) to produce CoA and 1,2-diacyl-sn-glycerol3-phosphate (phosphatidic acid). The literature also refers to LPAAT asacyl-CoA:1-acyl-sn-glycerol-3-phosphate 2-O-acyltransferase,1-acyl-sn-glycerol-3-phosphate acyltransferase and/or1-acylglycerolphosphate acyltransferase (abbreviated as AGAT).

The term “LPCAT” refers to an acyl-CoA:1-acyl lysophosphatidyl-cholineacyltransferase. This enzyme is responsible for the exchange of acylgroups between CoA and phosphatidyl choline (PC). Herein it also refersto enzymes involved the acyl exchange between CoA and otherphospholipids, including lysophosphatidic acid (LPA).

“Percent (%) PUFAs in the total lipid and oil fractions” refers to thepercent of PUFAs relative to the total fatty acids in those fractions.The term “total lipid fraction” or “lipid fraction” both refer to thesum of all lipids (i.e., neutral and polar) within an oleaginousorganism, thus including those lipids that are located in thephosphatidylcholine (PC) fraction, phosphatidyletanolamine (PE) fractionand triacylglycerol (TAG or oil) fraction. However, the terms “lipid”and “oil” will be used interchangeably throughout the specification.

The term “phosphatidylcholine” or “PC” refers to a phospholipid that isa major constituent of cell membranes. The chemical structure of PC cangenerally be described as comprising the following: a choline molecule,a phosphate group and glycerol, wherein fatty acyl chains are attachedas R groups on the sn-1 and sn-2 positions of the glycerol molecule.

The term “PUFA biosynthetic pathway enzyme” refers to any of thefollowing enzymes (and genes which encode said enzymes) associated withthe biosynthesis of a PUFA, including: a Δ4 desaturase, a Δ5 desaturase,a Δ6 desaturase, a Δ12 desaturase, a Δ15 desaturase, a Δ17 desaturase, aΔ9 desaturase, a Δ8 desaturase, a Δ9 elongase, a C_(14/16) elongase, aC_(16/18) elongase, a C_(18/20) elongase and/or a C_(20/22) elongase.The term “ω-3/ω-6 fatty acid biosynthetic pathway” refers to a set ofgenes which, when expressed under the appropriate conditions encodeenzymes that catalyze the production of either or both ω-3 and ω-6 fattyacids. Typically the genes involved in the ω-3/ω-6 fatty acidbiosynthetic pathway encode some or all of the following enzymes: Δ12desaturase, Δ6 desaturase, C_(18/20) elongase, C_(20/22) elongase, Δ9elongase, Δ5 desaturase, Δ17 desaturase, Δ15 desaturase, Δ9 desaturase,Δ8 desaturase and Δ4 desaturase. A representative pathway is illustratedin FIG. 1, providing for the conversion of oleic acid through variousintermediates to DHA, which demonstrates how both ω-3 and ω-6 fattyacids may be produced from a common source. The pathway is naturallydivided into two portions where one portion will generate ω-3 fattyacids and the other portion, only ω-6 fatty acids. That portion thatonly generates ω-3 fatty acids will be referred to herein as the ω-3fatty acid biosynthetic pathway, whereas that portion that generatesonly ω-6 fatty acids will be referred to herein as the ω-6 fatty acidbiosynthetic pathway.

The term “functional” as used herein in context with the ω-3/ω-6 fattyacid biosynthetic pathway means that some (or all of) the genes in thepathway express active enzymes, resulting in in vivo catalysis orsubstrate conversion. It should be understood that “ω-3/ω-6 fatty acidbiosynthetic pathway” or “functional ω-3/ω-6 fatty acid biosyntheticpathway” does not imply that all the genes listed in the above paragraphare required, as a number of fatty acid products will only require theexpression of a subset of the genes of this pathway.

The term “ω-6 Δ6 desaturase/Δ6 elongase pathway” will refer to a DHAfatty acid biosynthetic pathway that minimally includes the followinggenes: Δ6 desaturase, C_(18/20) elongase, Δ5 desaturase, Δ17 desaturase,C_(20/22) elongase and Δ4 desaturase. The term “ω-3 Δ6 desaturase/Δ6elongase pathway” will refer to a DHA fatty acid biosynthetic pathwaythat minimally includes the following genes: Δ15 desaturase, Δ6desaturase, C_(18/20)elongase, Δ5 desaturase, C_(20/22) elongase and Δ4desaturase. The term “combination Δ6 desaturase/Δ6 elongase pathway”will refer to a DHA fatty acid biosynthetic pathway that minimallyincludes the following genes: Δ15 desaturase, Δ6 desaturase,C_(18/20)elongase, Δ5 desaturase, Δ17 desaturase, C_(20/22) elongase andΔ4 desaturase. Finally, the term “Δ6 desaturase/Δ6 elongase pathway”will generically refer to any one (or more) of the Δ6 desaturase/Δ6elongase pathways described above.

In a related manner, the term “ω-6 Δ9 elongase/Δ8 desaturase pathway”will refer to a DHA fatty acid biosynthetic pathway that minimallyincludes the following genes: Δ9 elongase, Δ8 desaturase, Δ5 desaturase,Δ17 desaturase, C_(20/22) elongase and Δ4 desaturase. The term “ω-3 Δ9elongase/Δ8 desaturase pathway” will refer to a DHA fatty acidbiosynthetic pathway that minimally includes the following genes: Δ15desaturase, Δ9 elongase, Δ8 desaturase, Δ5 desaturase, C_(20/22)elongase and Δ4 desaturase. The term “combination Δ9 elongase/Δ8desaturase pathway” will refer to a DHA fatty acid biosynthetic pathwaythat minimally includes the following genes: Δ15 desaturase, Δ9elongase, Δ8 desaturase Δ5 desaturase, Δ17 desaturase, C_(20/22)elongase and Δ4 desaturase. And, the term “Δ9 elongase/Δ8 desaturasepathway” will generically refer to any one (or more) of the Δ9elongase/Δ8 desaturase pathways described above.

The term “desaturase” refers to a polypeptide that can desaturate, i.e.,introduce a double bond, in one or more fatty acids to produce a fattyacid or precursor of interest. Despite use of-the omega-reference systemthroughout the specification to refer to specific fatty acids, it ismore convenient to indicate the activity of a desaturase by countingfrom the carboxyl end of the substrate using the delta-system. Ofparticular interest herein are: 1.) Δ8 desaturases that desaturate afatty acid between the 8^(th) and 9^(th) carbon atom numbered from thecarboxyl-terminal end of the molecule and which, for example, catalyzethe conversion of EDA to DGLA and/or ETrA to ETA; 2.) Δ6 desaturasesthat catalyze the conversion of LA to GLA and/or ALA to STA; 3.) Δ5desaturases that catalyze the conversion of DGLA to ARA and/or ETA toEPA; 4.) Δ4 desaturases that catalyze the conversion of DPA to DHA; 5.)Δ12 desaturases that catalyze the conversion of oleic acid to LA; 6.)Δ15 desaturases that catalyze the conversion of LA to ALA and/or GLA toSTA; 7.) Δ17 desaturases that catalyze the conversion of ARA to EPAand/or DGLA to ETA; and 8.) Δ9 desaturases that catalyze the conversionof palmitate to palmitoleic acid (16:1) and/or stearate to oleic acid(18:1).

The term “bifunctional” as it refers to Δ15 desaturases of the inventionmeans that the polypeptide has the ability to use both oleic acid and LAas an enzymatic substrate. Similarly, the term “bifunctional” as itrefers to Δ5 desaturases of the invention means that the polypeptide hasthe ability to use: (1) at least one enzymatic substrate selected fromthe group consisting of DGLA and ETA; and (2) at least one enzymaticsubstrate selected from the group consisting of LA and ALA. By“enzymatic substrate” it is meant that the polypeptide binds thesubstrate at an active site and acts upon it in a reactive manner.

The term “elongase system” refers to a suite of four enzymes that areresponsible for elongation of a fatty acid carbon chain to produce afatty acid that is 2 carbons longer than the fatty acid substrate thatthe elongase system acts upon. More specifically, the process ofelongation occurs in association with fatty acid synthase, whereby CoAis the acyl carrier (Lassner et al., The Plant Cell 8:281-292 (1996)).In the first step, which has been found to be both substrate-specificand also rate-limiting, malonyl-CoA is condensed with a long-chainacyl-CoA to yield CO₂ and a β-ketoacyl-CoA (where the acyl moiety hasbeen elongated by two carbon atoms). Subsequent reactions includereduction to β-hydroxyacyl-CoA, dehydration to an enoyl-CoA and a secondreduction to yield the elongated acyl-CoA. Examples of reactionscatalyzed by elongase systems are the conversion of GLA to DGLA, STA toETA and EPA to DPA.

For the purposes herein, an enzyme catalyzing the first condensationreaction (i.e., conversion of malonyl-CoA to β-ketoacyl-CoA) will bereferred to generically as an “elongase”. In general, the substrateselectivity of elongases is somewhat broad but segregated by both chainlength and the degree and type of unsaturation. Accordingly, elongasescan have different specificities. For example, a C_(14/16) elongase willutilize a C₁₄ substrate (e.g., myristic acid), a C_(16/18) elongase willutilize a C₁₆ substrate (e.g., palmitate), a C_(18/20)elongase willutilize a C₁₈ substrate (e.g., GLA, STA) and a C_(20/22) elongase willutilize a C₂₀ substrate (e.g., EPA). In like manner, a Δ9 elongase isable to catalyze the conversion of LA and ALA to EDA and ETrA,respectively. It is important to note that some elongases have broadspecificity and thus a single enzyme may be capable of catalyzingseveral elongase reactions (e.g., thereby acting as both a C_(16/18)elongase and a C_(18/20) elongase). In preferred embodiments, it is mostdesirable to empirically determine the specificity of a fatty acidelongase by transforming a suitable host with the gene for the fattyacid elongase and determining its effect on the fatty acid profile ofthe host.

The term “high affinity elongase” or “EL1S” or “ELO1” refers to aC_(18/20) elongase whose substrate specificity is preferably for GLA(with DGLA as a product of the elongase reaction [i.e., a Δ6 elongase]).One such elongase is described in WO 00/12720 and is provided herein asSEQ ID NOs:22 and 23. However, the Applicants have shown that thisenzyme also has some activity on 18:2 (LA) and 18:3 (ALA); thus, SEQ IDNO:23 shows Δ9 elongase activity (in addition to its Δ6 elongaseactivity). It is therefore concluded that the C_(18/20) elongaseprovided herein as SEQ ID NO:23 can function both within the Δ6desaturase/Δ6 elongase pathway as described in the invention herein andwithin the Δ9 elongase/Δ8 desaturase pathway, as a substitute for e.g.,the Isochrysis galbana Δ9 elongase (SEQ ID NO:70).

The term “EL2S” or “ELO2” refers to a C_(18/20) elongase whose substratespecificity is preferably for GLA (with DGLA as a product of theelongase reaction) and/or STA (with STA as a product of the elongasereaction). One such elongase is described in U.S. Pat. No. 6,677,145 andis provided herein as SEQ ID NOs:25 and 26.

The term “ELO3” refers to a Mortierella alpina C_(16/18) fatty acidelongase enzyme (provided herein as SEQ ID NO:87), encoded by the elo3gene (SEQ ID NO:86). The term “YE2” refers to a Yarrowia lipolyticaC_(16/18) fatty acid elongase enzyme (provided herein as SEQ ID NO:95),encoded by the gene provided herein as SEQ ID NO:94. Based on datareported herein, both ELO3 amd YE2 preferentially catalyze theconversion of palmitate (16:0) to stearic acid (18:0).

The term “YE1” refers to a Yarrowia lipolytica C_(14/16) fatty acidelongase enzyme (provided herein as SEQ ID NO:98), encoded by the geneprovided herein as SEQ ID NO:97. Based on data reported herein, YE2preferentially catalyzes the conversion of myristic acid (14:0) topalmitate (16:0).

The terms “conversion efficiency” and “percent substrate conversion”refer to the efficiency by which a particular enzyme (e.g., a desaturaseor elongase) can convert substrate to product. The conversion efficiencyis measured according to the following formula:([product]/[substrate+product])*100, where ‘product’ includes theimmediate product and all products in the pathway derived from it.

The term “oleaginous” refers to those organisms that tend to store theirenergy source in the form of lipid (Weete, In: Fungal LipidBiochemistry, 2^(nd) Ed., Plenum, 1980). Generally, the cellular oilcontent of these microorganisms follows a sigmoid curve, wherein theconcentration of lipid increases until it reaches a maximum at the latelogarithmic or early stationary growth phase and then graduallydecreases during the late stationary and death phases (Yongmanitchai andWard, Appl. Environ. Microbiol. 57:419-25 (1991)).

The term “oleaginous yeast” refers to those microorganisms classified asyeasts that can make oil. Generally, the cellular oil or triacylglycerolcontent of oleaginous microorganisms follows a sigmoid curve, whereinthe concentration of lipid increases until it reaches a maximum at thelate logarithmic or early stationary growth phase and then graduallydecreases during the late stationary and death phases (Yongmanitchai andWard, Appl. Environ. Microbiol. 57:419-25 (1991)). It is not uncommonfor oleaginous microorganisms to accumulate in excess of about 25% oftheir dry cell weight as oil. Examples of oleaginous yeast include, butare no means limited to, the following genera: Yarrowia, Candida,Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

The term “fermentable carbon source” means a carbon source that amicroorganism will metabolize to derive energy. Typical carbon sourcesof the invention include, but are not limited to: monosaccharides,oligosaccharides, polysaccharides, alkanes, fatty acids, esters of fattyacids, monoglycerides, diglycerides, triglycerides, carbon dioxide,methanol, formaldehyde, formate and carbon-containing amines.

As used herein, an “isolated nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

A “substantial portion” of an amino acid or nucleotide sequence is thatportion comprising enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to putatively identify thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Basic Local AlignmentSearch Tool; Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1993)).In general, a sequence of ten or more contiguous amino acids or thirtyor more nucleotides is necessary in order to identify putatively apolypeptide or nucleic acid sequence as homologous to a known protein orgene. Moreover, with respect to nucleotide sequences, gene-specificoligonucleotide probes comprising 20-30 contiguous nucleotides may beused in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12-15 bases may be used as amplification primers inPCR in order to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises enough of the sequence to specifically identify and/or isolatea nucleic acid fragment comprising the sequence.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable of hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine.

“Codon degeneracy” refers to the nature in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. The skilled artisan is well aware ofthe “codon-bias” exhibited by a specific host cell in usage ofnucleotide codons to specify a given amino acid. Therefore, whensynthesizing a gene for improved expression in a host cell, it isdesirable to design the gene such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Chemically synthesized”, as related to a sequence of DNA, means thatthe component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established procedures;or, automated chemical synthesis can be performed using one of a numberof commercially available machines. “Synthetic genes” can be assembledfrom oligonucleotide building blocks that are chemically synthesizedusing procedures known to those skilled in the art. These buildingblocks are ligated and annealed to form gene segments that are thenenzymatically assembled to construct the entire gene. Accordingly, thegenes can be tailored for optimal gene expression based on optimizationof nucleotide sequence to reflect the codon bias of the host cell. Theskilled artisan appreciates the likelihood of successful gene expressionif codon usage is biased towards those codons favored by the host.Determination of preferred codons can be based on a survey of genesderived from the host cell, where sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, and that may refer to the coding region alone or may includeregulatory sequences preceding (5′ non-coding sequences) and following(3′ non-coding sequences) the coding sequence. “Native gene” refers to agene as found in nature with its own regulatory sequences. “Chimericgene” refers to any gene that is not a native gene, comprisingregulatory and coding sequences that are not found together in nature.Accordingly, a chimeric gene may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. “Endogenousgene” refers to a native gene in its natural location in the genome ofan organism. A “foreign” gene refers to a gene that is introduced intothe host organism by gene transfer. Foreign genes can comprise nativegenes inserted into a non-native organism, native genes introduced intoa new location within the native host, or chimeric genes. A “transgene”is a gene that has been introduced into the genome by a transformationprocedure. A “codon-optimized gene” is a gene having its frequency ofcodon usage designed to mimic the frequency of preferred codon usage ofthe host cell.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Suitable regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing sites, effector binding sites andstem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The term “GPAT promoter” or “GPAT promoter region” refers to the 5′upstream untranslated region in front of the ‘ATG’ translationinitiation codon of a glycerol-3-phosphate O-acyltransferase enzyme(E.C. 2.3.1.15) encoded by the gpat gene and that is necessary forexpression.

Examples of suitable Yarrowia lipolytica GPAT promoter regions aredescribed in U.S. patent application Ser. No. 11/225,354.

The term “GPD promoter” or “GPD promoter region” refers to the 5′upstream untranslated region in front of the ‘ATG’ translationinitiation codon of a glyceraldehyde-3-phosphate dehydrogenase enzyme(E.C. 1.2.1.12) encoded by the gpd gene and that is necessary forexpression. Examples of suitable Yarrowia lipolytica GPD promoterregions are described in WO 2005/003310.

The term “GPM promoter” or “GPM promoter region” refers to the 5′upstream untranslated region in front of the ‘ATG’ translationinitiation codon of a phosphoglycerate mutase enzyme (EC 5.4.2.1)encoded by the gpm gene and that is necessary for expression. Examplesof suitable Yarrowia lipolytica GPM promoter regions are described in WO2005/003310.

The term “FBA promoter” or “FBA promoter region” refers to the 5′upstream untranslated region in front of the ‘ATG’ translationinitiation codon of a fructose-bisphosphate aldolase enzyme (E.C.4.1.2.13) encoded by the fba1 gene and that is necessary for expression.Examples of suitable Yarrowia lipolytica FBA promoter regions aredescribed in WO 2005/049805.

The term “FBAIN promoter” or “FBAIN promoter region” refers to the 5′upstream untranslated region in front of the ‘ATG’ translationinitiation codon of the fba1 gene and that is necessary for expression,plus a portion of 5′ coding region that has an intron of the fba1 gene.Examples of suitable Yarrowia lipolytica FBAIN promoter regions aredescribed in WO 2005/049805.

The term “GPDIN promoter” or “GPDIN promoter region” refers to the 5′upstream untranslated region in front of the ‘ATG’ translationinitiation codon of the gpd gene and that is necessary for expression,plus a portion of 5′ coding region that has an intron of the gpd gene.Examples of suitable Yarrowia lipolytica GPDIN promoter regions aredescribed in U.S. patent application Ser. No. 11/183,664.

The term “YAT1 promoter” or “YAT1 promoter region” refers to the 5′upstream untranslated region in front of the ‘ATG’ translationinitiation codon of an ammonium transporter enzyme (TC 2.A.49; GenBankAccession No. XM_(—)504457) encoded by the yat1 gene and that isnecessary for expression. Examples of suitable Yarrowia lipolytica YAT1promoter regions are described in U.S. patent application Ser. No.11/185,301.

The term “EXP1 promoter” or “EXP1 promoter region” refers to the 5′upstream untranslated region in front of the ‘ATG’ translationinitiation codon of a protein encoded by the Yarrowia lipolytica“YALI0C12034g” gene (GenBank Accession No. XM_(—)501745) and that isnecessary for expression. Based on significant homology of“YALI0C12034g” to the sp|Q12207 S. cerevisiae non-classical exportprotein 2 (whose function is involved in a novel pathway of export ofproteins that lack a cleavable signal sequence), this gene is hereindesignated as the exp1 gene, encoding a protein designated as EXP1. Anexample of a suitable Yarrowia lipolytica EXP1 promoter region isdescribed as SEQ ID NO:221, but this is not intended to be limiting innature. One skilled in the art will recognize that since the exactboundaries of the EXP1 promoter sequence have not been completelydefined, DNA fragments of increased or diminished length may haveidentical promoter activity.

The term “promoter activity” will refer to an assessment of thetranscriptional efficiency of a promoter. This may, for instance, bedetermined directly by measurement of the amount of mRNA transcriptionfrom the promoter (e.g., by Northern blotting or primer extensionmethods) or indirectly by measuring the amount of gene product expressedfrom the promoter.

“Introns” are sequences of non-coding DNA found in gene sequences(either in the coding region, 5′ non-coding region, or 3′ non-codingregion) in most eukaryotes. Their full function is not known; however,some enhancers are located in introns (Giacopelli F. et al., Gene Expr.11: 95-104 (2003)). These intron sequences are transcribed, but removedfrom within the pre-mRNA transcript before the mRNA is translated into aprotein. This process of intron removal occurs by self-splicing of thesequences (exons) on either side of the intron.

The term “enhancer” refers to a cis-regulatory sequence that can elevatelevels of transcription from an adjacent eukaryotic promoter, therebyincreasing transcription of the gene. Enhancers can act on promotersover many tens of kilobases of DNA and can be 5′ or 3′ to the promoterthey regulate. Enhancers can also be located within introns.

The terms “3′ non-coding sequences” and “transcription terminator” referto DNA sequences located downstream of a coding sequence. This includespolyadenylation recognition sequences and other sequences encodingregulatory signals capable of affecting mRNA processing or geneexpression. The polyadenylation signal is usually characterized byaffecting the addition of polyadenylic acid tracts to the 3′ end of themRNA precursor. The 3′ region can influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA” or “mRNA” refersto the RNA that is without introns and that can be translated intoprotein by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to, and derived from, mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065; WO99/28508). The complementarity of an antisense RNA may be with any partof the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, or the coding sequence. “Functional RNA” refers toantisense RNA, ribozyme RNA, or other RNA that is not translated and yethas an effect on cellular processes.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragments of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be (but are not limited to)intracellular localization signals.

The term “recombinase” refers to an enzyme(s) that carries outsite-specific recombination to alter the DNA structure and includestransposases, lambda integration/excision enzymes, as well assite-specific recombinases.

“Recombinase site” or “site-specific recombinase sequence” means a DNAsequence that a recombinase will recognize and bind to. It will beappreciated that this may be a wild type or mutant recombinase site, aslong as functionality is maintained and the recombinase enzyme may stillrecognize the site, bind to the DNA sequence, and catalyze therecombination between two adjacent recombinase sites.

“Transformation” refers to the transfer of a nucleic acid molecule intoa host organism, resulting in genetically stable inheritance. Thenucleic acid molecule may be a plasmid that replicates autonomously, forexample; or, it may integrate into the genome of the host organism. Hostorganisms containing the transformed nucleic acid fragments are referredto as “transgenic” or “recombinant” or “transformed” organisms.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes that are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA fragments. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Expression cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that allow for enhanced expression of that gene in a foreign host.

The term “homologous recombination” refers to the exchange of DNAfragments between two DNA molecules (during cross over). The fragmentsthat are exchanged are flanked by sites of identical nucleotidesequences between the two DNA molecules (i.e., “regions of homology”).The term “regions of homology” refer to stretches of nucleotide sequenceon nucleic acid fragments that participate in homologous recombinationthat have homology to each other. Effective homologous recombinationwill generally take place where these regions of homology are at leastabout 10 bp in length where at least about 50 bp in length is preferred.Typically fragments that are intended for recombination contain at leasttwo regions of homology where targeted gene disruption or replacement isdesired.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1.) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.)Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized.

The term “conserved domain” or “motif” means a set of amino acidsconserved at specific positions along an aligned sequence ofevolutionarily related proteins. While amino acids at other positionscan vary between homologous proteins, amino acids that are highlyconserved at specific positions indicate amino acids that are essentialin the structure, the stability, or the activity of a protein. Becausethey are identified by their high degree of conservation in alignedsequences of a family of protein homologues, they can be used asidentifiers, or “signatures”, to determine if a protein with a newlydetermined sequence belongs to a previously identified protein family. Amotif that is indicative of a fungal protein having Δ15 desaturaseactivity is provided as SEQ ID NO:223, while a motif that is indicativeof a fungal protein having Δ12 desaturase activity is provided as SEQ IDNO:224.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989)(hereinafter “Maniatis”); by Silhavy, T. J., Bennan, M. L. and Enquist,L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory:Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience (1987).

A Preferred Microbial Host For DHA Production: Yarrowia lipolytica

Prior to work by the Applicants (see, Picataggio et al., WO2004/101757),oleaginous yeast have not been examined previously as a class ofmicroorganisms suitable for use as a production platform for PUFAs.Genera typically identified as oleaginous yeast include, but are notlimited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium,Cryptococcus, Trichosporon and Lipomyces. More specifically,illustrative oil-synthesizing yeast include: Rhodosporidium toruloides,Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C.tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorulaglutinus, R. graminis and Yarrowia lipolytica (formerly classified asCandida lipolytica).

Oleaginous yeast were considered to have several qualities that wouldfaciliate their use as a host organism for economical, commercialproduction of DHA. First, the organisms are defined as those that arenaturally capable of oil synthesis and accumulation, wherein the oil cancomprise greater than about 25% of the cellular dry weight, morepreferably greater than about 30% of the cellular dry weight and mostpreferably greater than about 40% of the cellular dry weight. Secondly,the technology for growing oleaginous yeast with high oil content iswell developed (for example, see EP 0 005 277B1; Ratledge, C., Prog.Ind. Microbiol. 16:119-206 (1982)). And, these organisms have beencommercially used for a variety of purposes in the past. For example,various strains of Yarrowia lipolytica have historically been used forthe manufacture and production of: isocitrate lyase (DD259637); lipases(SU1454852, WO2001083773, DD279267); polyhydroxyalkanoates(WO2001088144); citric acid (RU2096461, RU2090611, DD285372, DD285370,DD275480, DD227448, PL160027); erythritol (EP770683); 2-oxoglutaric acid(DD267999); γ-decalactone (U.S. Pat. No. 6,451,565, FR2734843);γ-dodecalactone (EP578388); and pyruvic acid (JP09252790).

Of those organisms classified as oleaginous yeast, Yarrowia lipolyticawas selected as the preferred microbial host for the purposes herein.This selection was based on the knowledge that oleaginous strains wereavailable that were capable of incorporating ω-3 fatty acids into theTAG fraction, the organism was amenable to genetic manipulation, andprevious use of the species as a Generally Recognized As Safe (“GRAS”,according to the U.S. Food and Drug Administration) source of food-gradecitric acid. In a further embodiment, most preferred are the Y.lipolytica strains designated as ATCC #20362, ATCC #8862, ATCC #18944,ATCC #76982 and/or LGAM S(7)1 (Papanikolaou S., and Aggelis G.,Bioresour. Technol. 82(1):43-9 (2002)), due to preliminary studiestargeted toward identification of wildtype strains having high lipidcontent (measured as a percent dry weight) and high volumetricproductivity (measured as g/L h⁻¹).

As described in WO 2004/101757, Yarrowia lipolytica was previouslygenetically engineered to produce 1.3% ARA and 1.9% EPA, respectively,by introduction and expression of genes encoding the ω-3/ω-6biosynthetic pathway. More specifically, two different DNA expressionconstructs (comprising either a Δ6 desaturase, Δ5 desaturase andhigh-affinity PUFA C_(18/20) elongase for ARA synthesis or a Δ6desaturase, Δ5 desaturase, high-affinity PUFA C_(18/20)elongase andcodon-optimized Δ17 desaturase for EPA synthesis) were separatelytransformed and integrated into the Y. lipolytica chromosomal URA3 geneencoding the enzyme orotidine-5′-phosphate decarboxylase (EC 4.1.1.23).GC analysis of the host cells fed with appropriate substrates detectedproduction of ARA and EPA. Although suitable to demonstrateproof-of-concept for the ability of oleaginous hosts to be geneticallyengineered for production of ω-6 and ω-3 fatty acids, this work failedto demonstrate production of DHA or suggest or perform the complexmetabolic engineering required to enable synthesis of greater than 5%DHA in the total oil fraction, or more preferably greater than 10% DHAin the total oil fraction, or even more preferably greater than 15-20%DHA in the total oil fraction, or most preferably greater than 25-30%DHA in the total oil fraction.

In co-pending U.S. Patent Application No. 60/624,812, complex metabolicengineering within Yarrowia lipolytica was performed to: (1) identifypreferred desaturases and elongases that allow for the synthesis andhigh accumulation of EPA; (2) manipulate the activity ofacyltransferases that allow for the transfer of omega fatty acids intostorage lipid pools; (3) over-express desaturases, elongases andacyltransferases by use of strong promoters, expression in multicopy,and/or codon-optimization; (4) down-regulate the expression of specificgenes within the PUFA biosynthetic pathway that diminish overallaccumulation of EPA; and, (5) manipulate pathways and global regulatorsthat affect EPA production. This resulted in the production of up to 28%EPA in one particular recombinant strain of Yarrowia lipolytica.

In the present Application, analogous complex metabolic engineering isperformed to result in the production of greater than 5% DHA in thetotal oil fraction in recombinant strains of Yarrowia lipolytica. Morespecifically, strains were genetically engineered to utilize the Δ6desaturase/Δ6 elongase pathway; in alternate embodiments, transformantstrains could be genetically engineered to utilize the Δ9 elongase/Δ8desaturase pathway and thereby produce a high-DHA oil that is devoid ofGLA. Aspects of the metabolic engineering utilized will be discussedbelow, as will additional engineering and fermentation methods thatcould be performed to significantly enhance DHA productivity in thisoleaginous yeast.

An Overview: Microbial Biosynthesis Of Fatty Acids and Triacylglycerols

In general, lipid accumulation in oleaginous microorganisms is triggeredin response to the overall carbon to nitrogen ratio present in thegrowth medium. This process, leading to the de novo synthesis of freepalmitate (16:0).in oleaginous microorganisms, is described in detail inWO 2004/101757. Palmitate is the precursor of longer-chain saturated andunsaturated fatty acid derivates, which are formed through the action ofelongases and desaturases. For example, palmitate is converted to itsunsaturated derivative [palmitoleic acid (16:1)] by the action of a Δ9desaturase; similarly, palmitate is elongated by a C_(16/18) fatty acidelongase to form stearic acid (18:0), which can be converted to itsunsaturated derivative by a Δ9 desaturase to thereby yield oleic (18:1)acid.

TAGs (the primary storage unit for fatty acids) are formed by a seriesof reactions that involve: 1.) the esterification of one molecule ofacyl-CoA to glycerol-3-phosphate via an acyltransferase to producelysophosphatidic acid; 2.) the esterification of a second molecule ofacyl-CoA via an acyltransferase to yield 1,2-diacylglycerol phosphate(commonly identified as phosphatidic acid); 3.) removal of a phosphateby phosphatidic acid phosphatase to yield 1,2-diacylglycerol (DAG); and4.) the addition of a third fatty acid by the action of anacyltransferase to form TAG (FIG. 2).

A wide spectrum of fatty acids can be incorporated into TAGs, includingsaturated and unsaturated fatty acids and short-chain and long-chainfatty acids. Some non-limiting examples of fatty acids that can beincorporated into TAGs by acyltransferases include: capric (10:0),lauric (12:0), myristic (14:0), palmitic (16:0), palmitoleic (16:1),stearic (18:0), oleic (18:1), vaccenic (18:1), LA, eleostearic (18:3),ALA, GLA, arachidic (20:0), EDA, ETrA, DGLA, ETA, ARA, EPA, behenic(22:0), DPA, DHA, lignoceric (24:0), nervonic (24:1), cerotic (26:0) andmontanic (28:0) fatty acids. In preferred embodiments of the presentinvention, incorporation of DHA into TAG is most desirable.

Biosynthesis of DHA, an ω-3 Fatty Acid

The metabolic process wherein oleic acid is converted to DHA involveselongation of the carbon chain through the addition of carbon atoms anddesaturation of the molecule through the addition of double bonds. Thisrequires a series of special desaturation and elongation enzymes presentin the endoplasmic reticulim membrane. However, as seen in FIG. 1 and asdescribed below, multiple alternate pathways exist for DHA production(although in all cases, production of DHA requires the synthesis ofEPA).

Specifically, all pathways require the initial conversion of oleic acidto LA (18:2), the first of the ω-6 fatty acids, by the action of a Δ12desaturase. Then, using the “ω-6 Δ6 desaturase/Δ6 elongase pathway” forEPA biosynthesis (whereby EPA biosynthesis occurs primarily through theformation of ω-6 fatty acids), PUFAs are formed as follows: (1) LA isconverted to GLA by the action of a Δ6 desaturase; (2) GLA is convertedto DGLA by the action of a C_(18/20) elongase; (3) DGLA is converted toARA by the action of a Δ5 desaturase; and (4) ARA is converted to EPA bythe action of a Δ17 desaturase. Alternatively, when EPA biosynthesisoccurs primarily through the formation of ω-3 fatty acids via the “ω-3Δ6 desaturase/Δ6 elongase pathway”, (1) LA is converted to ALA, thefirst of the ω-3 fatty acids, by the action of a Δ15 desaturase; (2) ALAis converted to STA by the action of a Δ6 desaturase; (3) STA isconverted to ETA by the action of a C_(18/20) elongase; and (4) ETA isconverted to EPA by the action of a Δ5 desaturase. Optionally, acombination of ω-6 and ω-3 fatty acids can be synthesized prior toproduction of EPA, either when ETA is produced from DGLA by the actionof a Δ17 desaturase, or when both Δ15 desaturase and Δ17 desaturase areco-expressed in conjunction with a Δ6 desaturase, C_(18/20) elongase andΔ5 desaturase.

Alternate pathways for the biosynthesis of EPA utilize a Δ9 elongase andΔ8 desaturase. More specifically, via the “ω-6 Δ9 elongase/Δ8 desaturasepathway”, LA is converted to EDA by the action of a Δ9 elongase; then, aΔ8 desaturase converts EDA to DGLA. Subsequent desaturation of DGLA bythe action of a Δ5 desaturase yields ARA, as described above, whereinARA can be converted directly to EPA by the action of a Δ17 desaturase.In contrast, using the “ω-3 Δ9 elongase/Δ8 desaturase pathway”, LA isfirst converted to ALA by the action of a Δ15 desaturase Then, ALA isconverted to ETrA by the action of a Δ9 elongase, followed by a Δ8desaturase that converts ETrA to ETA. Subsequent desaturation of ETA bythe action of a Δ5 desaturase yields EPA.

Upon synthesis of EPA, a C_(20/22) elongase is responsible forconversion of the substrate to DPA. Then, DPA is converted to DHA by theaction of a Δ4 desaturase.

For the sake of clarity, each of these pathways will be summarized inthe Table below, as well as their distinguishing characteristics:

TABLE 4 Alternate Biosynthetic Pathways For DHA Biosynthesis MinimumRequired Genes Name For DHA* Pathway ω-6 Δ6 desaturase/ Δ6D, C_(18/20)improves the ω-3/ω-6 ratio of Δ6 elongase ELO, Δ5D, PUFA productspathway Δ17D, C_(20/22) ELO, Δ4D ω-3 Δ6 desaturase/ Δ15D, Δ6D, improvesthe ω-3/ω-6 ratio of Δ6 elongase C_(18/20) ELO, substrates forsubsequent pathway Δ5D, C_(20/22) PUFA biosynthesis; produces oil ELO,Δ4D that is devoid of GLA Combination Δ6 Δ15D, Δ6D, — desaturase/Δ6C_(18/20) ELO, elongase pathway Δ5D, Δ17D, C_(20/22) ELO, Δ4D ω-6 Δ9elongase/Δ8 Δ9 ELO, improves the ω-3/ω-6 ratio of desaturase pathwayΔ8D, Δ5D, PUFA products Δ17D, C_(20/22) ELO, Δ4D ω-3 Δ9 elongase/Δ8Δ15D, Δ9 ELO, improves the ω-3/ω-6 ratio of desaturase pathway Δ8D, Δ5D,C_(20/22) substrates for subsequent ELO, Δ4D PUFA biosynthesis; producesoil that is devoid of GLA Combination Δ9 Δ15D, Δ9 ELO, — elongase/Δ8Δ8D, Δ5D, desaturase pathway Δ17D, C_(20/22) ELO, Δ4D *Abbreviations:“D” = desaturase; “ELO” = elongase.

Selection of Microbial Genes for DHA Synthesis

The particular functionalities required to be introduced into Yarrowialipolytica for production of DHA will depend on the host cell (and itsnative PUFA profile and/or desaturase/elongase profile), theavailability of substrate, and the desired end product(s). With respectto the native host cell, it is known that Y. lipolytica can naturallyproduce 18:2 fatty acids and thus possesses a native Δ12 desaturase (SEQID NOs:28 and 29; see WO 2004/104167). With respect to the desired endproducts, the consequences of Δ6 desaturase/Δ6 elongase pathwayexpression as opposed to Δ9 elongase/Δ8 desaturase pathway expressionhave been described above, in terms of the final fatty acid profile ofoil so produced (i.e., % GLA in the final composition of high DHA oil).

In some embodiments, it will therefore be desirable to produce DHA viathe Δ6 desaturase/Δ6 elongase pathway. Thus, at a minimum, the followinggenes must be introduced into the host organism and expressed for DHAbiosythesis: a Δ6 desaturase, a C_(18/20) elongase, a Δ5 desaturase,either a Δ17 desaturase or a Δ15 desaturase (or both), a C_(20/22)elongase and a Δ4 desaturase. In a further preferred embodiment, thehost strain additionally includes at least one of the following: a Δ9desaturase, a Δ12 desaturase, a C_(14/16) elongase and a C_(16/18)elongase.

In alternate embodiments, it is desirable to produce DHA withoutco-synthesis of GLA (thus requiring expression of the Δ9 elongase/Δ8desaturase pathway). This strategy thereby minimally requires thefollowing genes to be introduced into the host organism and expressedfor DHA biosythesis: a Δ9 elongase, a Δ8 desaturase, a Δ5 desaturase,either a Δ17 desaturase or a Δ15 desaturase (or both), a C_(20/22)elongase and a Δ4 desaturase. In a further preferred embodiment, thehost strain additionally includes at least one of the following: a Δ9desaturase, a Δ12 desaturase, a C_(14/16) elongase and a C_(16/18)elongase.

One skilled in the art will be able to identify various candidate genesencoding each of the enzymes desired for DHA biosynthesis. Usefuldesaturase and elongase sequences may be derived from any source, e.g.,isolated from a natural source (from bacteria, algae, fungi, plants,animals, etc.), produced via a semi-synthetic route or synthesized denovo. Although the particular source of the desaturase and elongasegenes introduced into the host is not critical to the invention,considerations for choosing a specific polypeptide having desaturase orelongase activity include: 1.) the substrate specificity of thepolypeptide; 2.) whether the polypeptide or a component thereof is arate-limiting enzyme; 3.) whether the desaturase or elongase isessential for synthesis of a desired PUFA; and/or 4.) co-factorsrequired by the polypeptide. The expressed polypeptide preferably hasparameters compatible with the biochemical environment of its locationin the host cell. For example, the polypeptide may have to compete forsubstrate with other enzymes in the host cell. Analyses of the K_(M) andspecific activity of the polypeptide are therefore considered indetermining the suitability of a given polypeptide for modifying PUFAproduction in a given host cell. The polypeptide used in a particularhost cell is one that can function under the biochemical conditionspresent in the intended host cell but otherwise can be any polypeptidehaving desaturase or elongase activity capable of modifying the desiredPUFA.

In additional embodiments, it will also be useful to consider theconversion efficiency of each particular desaturase and/or elongase.More specifically, since each enzyme rarely functions with 100%efficiency to convert substrate to product, the final lipid profile ofun-purified oils produced in a host cell will typically be a mixture ofvarious PUFAs consisting of the desired DHA, as well as various upstreamintermediary PUFAs (e.g., as opposed to 100% DHA oil). Thus,consideration of each enzyme's conversion efficiency is also animportant variable when optimizing biosynthesis of DHA, that must beconsidered in light of the final desired lipid profile of the product.

With each of the considerations above in mind, candidate genes havingthe appropriate desaturase and elongase activities can be identifiedaccording to publicly available literature (e.g., GenBank), the patentliterature, and experimental analysis of microorganisms having theability to produce PUFAs. For instance, the following GenBank AccessionNumbers refer to examples of publicly available genes useful in DHAbiosynthesis: AY131238, Y055118, AY055117, AF296076, AF007561, L11421,NM_(—)031344, AF465283, AF465281, AF110510, AF465282, AF419296,AB052086, AJ250735, AF126799, AF126798 (Δ6 desaturases); AF390174 (Δ9elongase); AF139720 (Δ8 desaturase); AF199596, AF226273, AF320509,AB072976, AF489588, AJ510244, AF419297, AF07879, AF067654, AB022097 (Δ5desaturases); AAG36933, AF110509, AB020033, AAL13300, AF417244,AF161219, AY332747, AAG36933, AF110509, AB020033, AAL13300, AF417244,AF161219, X86736, AF240777, AB007640, AB075526, AP002063 (Δ12desaturases); NP_(—)441622, BAA1 8302, BAA02924, AAL36934 (Δ15desaturases); AF338466, AF438199, E11368, E11367, D83185, U90417,AF085500, AY504633, NM_(—)069854, AF230693 (Δ9 desaturases); AY630574,AY332747, AY278558, AF489589 (Δ4 desaturases); and NP_(—)012339,NP_(—)009963, NP_(—)013476, NP_(—)599209, BAB69888, AF244356, AAF70417,AAF71789, AF390174, AF428243, NP_(—)955826, AF206662, AF268031,AY591335, AY591336, AY591337, AY591338, AY605098, AY605100, AY630573(C_(14/16), C_(16/18), C_(18/20), and C_(20/22) elongases). Similarly,the patent literature provides many additional DNA sequences of genes(and/or details concerning several of the genes above and their methodsof isolation) involved in PUFA production [e.g., WO 02/077213 (Δ9elongases); WO 00/34439 and WO 04/057001 (Δ8 desaturases); U.S. Pat. No.5,968,809 (Δ6 desaturases); U.S. Pat. No. 5,972,664 and U.S. Pat. No.6,075,183 (Δ5 desaturases); WO 94/11516, U.S. Pat. No. 5,443,974, WO03/099216 and WO 05/047485 (Δ12 desaturases); WO 93/11245 (Δ15desaturases); WO 91/13972 and U.S. Pat. No. 5,057,419 (Δ9 desaturases);U.S. 2003/0196217 (Δ17 desaturases); WO 02/090493 (Δ4 desaturases); and,WO 00/12720, U.S. Pat. No. 6,403,349, U.S. Pat. No. 6,677,145, U.S.2002/0139974A1, U.S. 2004/0111763 (C_(14/16), C_(16/18), C_(18/20), andC_(20/22) elongases)]. Each of these patents and applications are hereinincorporated by reference in their entirety.

The examples above are not intended to be limiting and numerous othergenes encoding (1) Δ6 desaturases, C_(18/20) elongases, Δ5 desaturases,either Δ17 desaturases or Δ15 desaturases (or both), C_(20/22) elongasesand Δ4 desaturases (and optionally other genes encoding Δ9 desaturases,Δ12 desaturases, C_(14/16) elongases and/or C_(16/18) elongases); or (2)Δ9 elongases, Δ8 desaturases, Δ5 desaturases, either Δ17 desaturases orΔ15 desaturases (or both), C_(20/22) elongases and Δ4 desaturases (andoptionally other genes encoding Δ9 desaturases, Δ12 desaturases,C_(14/16) elongases and/or C_(16/18) elongases) derived from differentsources would be suitable for introduction into Yarrowia lipolytica.

Preferred Genes for DHA Synthesis

Despite the wide selection of desaturases and elongases that could besuitable for expression in Yarrowia lipolytica, however, in preferredembodiments of the present invention the desaturases and elongases areselected from the following (or derivatives thereof):

TABLE 5 Preferred Desaturases And Elongases For DHA Biosynthesis InYarrowia lipolytica SEQ ID ORF Organism Reference NOs Δ6 MortierellaGenBank Accession No. 1, 2 desaturase alpina AF465281; U.S. Pat. No.5,968,809 Δ6 Mortierella GenBank Accession No. 4, 5 desaturase alpinaAB070555 C_(18/20) Mortierella GenBank Accession No. 22, elongase alpinaAX464731; WO 00/12720 23 (“ELO1”) C_(18/20) Thrausto- U.S. Pat. No.6,677,145 25, elongase chytrium 26 (“ELO2”) aureum Δ9 elongaseIsochrysis GenBank Accession No. 69, galbana AF390174 70 Δ8 Euglenagracillis Co-pending U.S. Patent 77, desaturase Application Number11/166993 78 Δ5 Mortierella GenBank Accession No. 6, 7 desaturase alpinaAF067654; U.S. Pat. No. 6,075,183 Δ5 Isochrysis WO 02/081668 A2 8, 9desaturase galbana Δ5 Homo sapiens GenBank Accession No. 11, desaturaseNP_037534 12 Δ5/Δ6 Danio rerio GenBank Accession No. 14, desaturaseAF309556 15 Δ5/Δ6 Danio rerio GenBank Accession No. 16 desaturaseBC068224 Δ5/Δ6 Danio rerio — 17, desaturase 18 Δ17 Saprolegnia US2003/0196217 A1 19, desaturase diclina 20 Δ4 Thrausto- GenBank AccessionNo. 104, desaturase chytrium AAN75707 105 aureum C_(20/22) OstreococcusGenBank Accession No. 100, elongase tauri AY591336 101 C_(16/18)Yarrowia — 94, elongase lipolytica 95 (“YE2”) C_(16/18) Mortierella —86, elongase alpina 87 (“ELO3”) C_(16/18) Rattus GenBank Accession No.83, elongase norvegicus AB071986 84 (rELO2) C_(14/16) Yarrowia — 97,elongase lipolytica 98 (“YE1”) Δ12 Yarrowia WO 2004/104167 28,desaturase lipolytica 29 Δ12 Mortieralla GenBank Accession No. 30,desaturase isabellina AF417245 31 Δ12 Fusarium WO 2005/047485 32,desaturase moniliforme 33 (Fm d12) Δ12 Aspergillus Contig 1.15(scaffold 1) in the A. nidulans 34, desaturase nidulans genome project;35 (An d12) AAG36933; WO 2005/047485 Δ12 Aspergillus GenBank AccessionNo. 36 desaturase flavus AY280867 (VERSION AY280867.1; gi: 30721844); WO2005/047485 Δ12 Aspergillus AFA.133c 344248:345586 37 desaturasefumigatus reverse (AfA5C5.001c) in the (Afd12p) Aspergillus fumigatusgenome project; WO 2005/047485 Δ12 Magnaporthe Locus MG01985.1 in contig38, desaturase grisea 2.375 in the M. grisea genome 39 (Mg d12) project;WO 2005/047485 Δ12 Neurospora GenBank Accession No. 40, desaturasecrassa AABX01000374; 41 (Nc d12) WO 2005/047485 Δ12 Fusarium Contig1.233 in the F. graminearium 42, desaturase graminearium genome project;43 (Fg d12) WO 2005/047485 Δ12 Mortierella GenBank Accession No. 44,desaturase alpina AB020033 45 (Mad12) Δ12 Saccharomyces GenBankAccession No. 46 desaturase kluyveri BAD08375 (Skd12) Δ12 Kluyveromycesgnl|GLV|KLLA0B00473g ORF 47, desaturase lactis from Klla0B: 35614 . . .36861 48 (Kld12p) antisense (m) of K. lactis database of the “Yeastproject Genolevures” (Center for Bioinformatics, LaBRI, Talence Cedex,France) Δ12 Candida GenBank Accession No. 49 desaturase albicansEAK94955 (Cad12p) Δ12 Debaryomyces GenBank Accession No. 50 desaturasehansenii CAG90237 (Dhd12p) CBS767 Δ15 Fusarium WO 2005/047479 51,desaturase moniliforme 52 (Fm d15) Δ15 Aspergillus Contig 1.122(scaffold 9) in the A. nidulans 53, desaturase nidulans genome project;54 (An d15) WO 2005/047479 Δ15 Magnaporthe Locus MG08474.1 in contig 55,desaturase grisea 2.1597 in the M. grisea genome 56 (Mg d15) project; WO2005/047479 Δ15 Neurospora GenBank Accession No. 57, desaturase crassaAABX01000577; 58 (Nc d15) WO 2005/047479 Δ15 Fusarium Contig 1.320 inthe F. graminearium 59, desaturase graminearium genome project 60 (Fgd15) (BAA33772.1); WO 2005/047479 Δ15 Mortierella GenBank Accession No.61, desaturase alpina AB182163 62 (Mad15) Δ15 Kluyveromyces GenBankAccession No. 63, desaturase lactis XM_451551 64 (Kld15p) Δ15 CandidaGenBank Accession No. 65 desaturase albicans EAL03493 (Cad15p) Δ15Saccharomyces GenBank Accession No. 66 desaturase kluyveri BAD11952(Skd15) Δ15 Debaryomyces GenBank Accession No. 67 desaturase hanseniiCAG88182 (Dhd15p) CBS767 Δ15 Aspergillus GenBank Accession No. 68desaturase fumigatus EAL85733 (Afd15p) * Note: The Aspergillus fumigatusgenome project is sponsored by Sanger Institute, collaborators at theUniversity of Manchester and The Institute of Genome Research (TIGR);the A. nidulans genome project is sponsored by the Center for GenomeResearch (CGR), Cambridge, MA; the M. grisea genome project is sponsoredby the CGR and International Rice Blast Genome Consortium; the F.graminearium genome project is sponsored by the CGR and theInternational Gibberella zeae Genomics Consortium (IGGR).

The Applicants have performed considerable analysis of variouselongases, to either determine or confirm each enzyme's substratespecificity and/or substrate selectivity when expressed in Yarrowialipolytica. For example, although the coding sequences of the two Y.lipolytica elongases were publically available and each protein wasannotated as a putative long-chain fatty-acyl elongase or sharedsignificant homology to other fatty acid elongases, the substratespecificity of these enzymes had never been determined. Based on theanalyses performed herein, YE1 was positively determined to be a fattyacid elongase that preferentially used C₁₄ fatty acids as substrates toproduce C₁₆ fatty acids (i.e., a C_(14/16) elongase) and YE2 wasdetermined to be a fatty acid elongase that preferentially used C₁₆fatty acids as substrates to produce C₁₈ fatty acids (i.e., a C_(16/18)elongase). Relatedly, upon identification of the novel M. alpina ELO3gene, the sequence was characterized as homologous to other fatty acidelongases; however, lipid profile analyses were required to confirm thespecificity of ELO3 as a C_(16/18) elongase.

With respect to Δ12 desaturase, the Applicants have made the surprisingdiscovery that the Fusarium moniliforme Δ12 desaturase (encoded by SEQID NO:32) functions with greater efficiency than the native Yarrowialipolytica Δ12 desaturase in producing 18:2 in Y. lipolytica (see WO2005/047485). Specifically, expression of the F. moniliforme Δ12desaturase under the control of the TEF promoter in Y. lipolytica wasdetermined to produce higher levels of 18:2 (68% product accumulation ofLA) than were previously attainable by expression of a chimeric geneencoding the Y. lipolytica Δ12 desaturase under the control of the TEFpromoter (59% product accumulation of LA). This corresponds to adifference in percent substrate conversion (calculated as([18:2+18:3]/[18:1+18:2+18:3])*100) of 85% versus 74%, respectively. Onthe basis of these results, expression of the present fungal F.moniliforme Δ12 desaturase is preferred relative to other known Δ12desaturases as a means to engineer a high DHA-producing strain of Y.lipolytica (however, one skilled in the art would expect that theactivity of the F. moniliforme Δ12 desaturase could be enhanced in Y.lipolytica, following e.g., codon-optimization).

Alternatively, five new Δ12 desaturases have recently been identifiedthat could possibly function with improved efficiency in Yarrowialipolytica. Specifically, the Saccharomyces kluyveri Δ12 desaturase(GenBank Accession No. BAD08375) was described in Watanabe et al.(Biosci. Biotech. Biocheml. 68(3):721-727 (2004)), while that fromMortierella alpina (GenBank Accession No. AB182163) was described bySakuradani et al. (Eur. J. Biochem. 261(3):812-820 (1999)). Using thesesequences, and the methodology described infra, three additional Δ12desaturases were identified by the Applicants herein: Kluyveromyceslactis gnl|GLV|KLLA0B00473g ORF (SEQ ID NO:48), Candida albicans GenBankAccession No. EAK94955 (SEQ ID NO:49) and Debaryomyces hansenii CBS767GenBank Accession No. CAG90237 (SEQ ID NO:50). Overexpression of any ofthese additional Δ12 desaturases in Yarrowia lipolytica could be usefulas a means to increase production of LA, thereby enabling increasedproduction of other downstream PUFAs (e.g., DHA).

In another preferred embodiment, F. moniliforme (SEQ ID NOs:51 and 52)is the preferred Δ15 desaturase for increasing the production of ALA,since this particular Δ15 desaturase possesses several uniquecharacteristics as compared to previously known Δ15 desaturases. First,the F. moniliforme Δ15 desaturase is distinguished by its significantΔ12 desaturase activity (thus characterizing the enzyme asbifunctional). Previous studies have determined that a Δ12desaturase-disrupted strain of Yarrowia lipolytica that was transformedwith a chimeric gene encoding SEQ ID NO:52 was able to convert 24% ofoleic acid to LA (percent substrate conversion calculated as([18:2+18:3]/[18:1+18:2+18:3])*100), in addition to 96% of LA to ALA(percent substrate conversion calculated as [18:3]/[18:2+18:3]*100)).Secondly, the F. moniliforme Δ15 desaturase enables very high synthesisof ALA when expressed in Y. lipolytica [i.e., Y. lipolytica that wastransformed with a chimeric gene encoding SEQ ID NO:52 was able todemonstrate a % product accumulation of ALA of 31%, relative to thetotal fatty acids in the transformant host cell, which is equivalent toa conversion efficiency to ALA of 83% (calculated as[18:3]/[18:2+18:3]*100)], relative to that described for otherheterologously expressed Δ15 desaturases (e.g., the % productaccumulation of ALA when expressing the C. elegans Δ15 desaturase in thenon-oleaginous yeast Sacchromyces cerevisiae was only 4.1% (Meesapyodsuket al., Biochem. 39:11948-11954 (2000)), while the % productaccumulation of ALA when expressing the B. napus Δ15 desaturase in S.cerevisiae was only 1.3% (Reed., D. W. et al., Plant Physiol.122:715-720 (2000)). Finally, the F. moniliforme Δ15 enzyme hasrelatively broad substrate specificity on downstream ω-6 derivatives of18:2; specifically, the Δ15 desaturase is able to catalyze conversion ofGLA to STA, DGLA to ETA, and ARA to EPA.

Despite the current identification of the F. moniliforme Δ15 enzyme asthe preferred Δ15 desaturase, six new Δ15 desaturases have recently beenidentified that could possibly function with improved efficiency inYarrowia lipolytica. Specifically, the Saccharomyces kluyveri Δ15desaturase (GenBank Accession No. BAD11952; Skd15) was described in Ouraet al. (Microbiol. 150:1983-1990 (2004)), while that from Mortierellaalpina (GenBank Accession No. AB182163; Mad15) was described bySakuradani et al. (Appl. Microbiol. Biotechnol. 66:648-654 (2005)).Since both sequences were identified in part based on their closehomology to previously identified S. kluyveri and M. alpina Δ12desaturases, respectively, followed by a determination of theirfunctional activity, these two pairs of proteins provided additionalexamples of closely related fungal Δ12 and Δ15 desaturases similar tothose of Fusarium moniliforme, Aspergillus nidulans, Magnaporthe grisea,Neurospora crassa and Fusarium graminearium (see Table above). Thisfinding offered additional support to the Applicants' previoushypothesis that “pairs” of fungal Δ12 desaturase-like sequences likelycomprise one protein having Δ15 desaturase activity and one proteinhaving Δ12 desaturase activity (see WO 2005/047480 and WO 2005/047485).Similar “pairs” of Δ12 desaturase-like proteins were thus identifiedherein in Kluyveromyces lactis, Candida albicans, Debaryomyces hanseniiCBS767 and Aspergillus fumigatus; and, as predicted, one member of eachpair aligned more closely to the previously identified S. kluyveri Δ12desaturase (Skd12) and the other more closely to Skd15 (FIG. 3A). Thus,based on this analysis, the Applicants have identified K. lactis GenBankAccession No. XM_(—)451551, D. hansenii CBS767 GenBank Accession No.CAG88182, C. albicans GenBank Accession No. EAL03493 and A. fumigatusGenBank Accession No. EAL85733 as putative fungal Δ15 desaturases whoseoverexpression in Y. lipolytica could be useful to increase productionof ω-3 fatty acids.

In additional embodiments, the Applicants have identified a means toreadily distinguish fungal sequences having Δ15 desaturase activity asopposed to Δ12 desaturase activity. Specifically, when an amino acidsequence alignment was analysed that comprised Mad12, Skd12, Nc d12, Fmd12, Mg d12, An d12, Fg d12, Dhd12p, Kld12p, Cad12p, Afd12p, Mad15,Skd15, Nc d15, Fm d15, Mg d15, An d15, Fg d15, Dhd15p, Kld15p, Cad15pand Afd15p (see Table above), it became apparent that all of the fungalΔ15 or Δ12 desaturases contained either an lie or Val amino acidresidue, respectively, at the position that corresponds to position 102of Fm d15 (SEQ ID NO:52) and that is only three amino acid residues awayfrom the highly conserved His Box 1 (“HECGH”; SEQ ID NO:222) (Table 6).

TABLE 6 Amino Acid Alignment Around The Conserved His Box 1 Of FungalΔ12 And Δ15 Desaturases

The Applicants conclude that Ile and Val at this position is adeterminant of Δ15 and Δ12 desaturase specificity, respectively, infungal desaturases. More specifically, the Applicants propose that anyfungal Δ12 desaturase-like protein with lie at the correspondingresidue(s) (i.e., or the motif IXXHECGH [SEQ ID NO:223]) will becharacterized as a Δ15 desaturase and any fungal Δ12 desaturase-likeprotein with Val at the corresponding residue(s) (i.e., or the motifVXXHECGH [SEQ ID NO:224]) will be characterized as a Δ12 desaturase.Thus, this single leucine/valine amino acid will be an important residueto consider as future fungal desaturases are identified and annotated.Futhermore, it is contemplated that mutation(s) that result in aIle-to-Val change at this position will alter enzyme specificity, suchas towards Δ12 desaturation, in genes encoding fungal Δ12desaturase-like proteins (e.g., the Fusarium monoliforme desaturasedescribed herein as SEQ ID NO: 52); and, conversely, those mutationsthat result in a Val-to-Ile change at this position will alter enzymespecificity, such as towards Δ15 desaturation.

In preferred embodiments various Δ5 desaturases may be selected as mostadvantageous to express in a host cell for DHA production, depending onthe particular pathway that is to be utilized. Specifically, whenexpressing the ω-6 Δ6 desaturase/Δ6 elongase pathway or the ω-6 Δ9elongase/Δ8 desaturase pathway, the M. alpina, I. galbana and H. sapiensΔ5 desaturases are preferred. In contrast, when it is desirable toutilize the ω-3 Δ6 desaturase/Δ6 elongase pathway or the ω-3 Δ9elongase/Δ8 desaturase pathway (thereby favoring synthesis of ω-3PUFAs), it may be advantageous to utilize an ω-3-preferring Δ5desaturase, such as that from Phytopthera megasperma or from Daniorerio. Hastings et al. originally reported that expression of a Daniorerio cDNA (GenBank Accession No. AF309556) in Saccharomyces cerevisiaeshowed bifunctional Δ6 and Δ5 desaturase activity with a distinctpreference for ω-3 compared with ω-6 substrates and slightly higher Δ6than Δ5 desaturase activity. Subsequently, the Applicants identifiedGenBank Accession No. BC068224 as a homolog of GenBank Accession No.AF309556, that differed only by a 1 bp (T) deletion at position 984 ofthe ORF (resulting in a null mutation) and a 1 bp change (G to A) atposition 1171 (resulting in a V to M amino acid change). A mutantprotein was then created (identified herein as “Drd6/d5(M)”) identicalto GenBank Access.ion No. AF309556, with the exception of the V1171 Mmutation of GenBank Accession No. BC068224. Although preliminary studiesby the Applicants herein determined that expression of Drd6/d5(M) in S.cerevisiae showed about 50% less activity than GenBank Accession No.AF309556, expression in a Yarrowia strain making ETA confirmed that thebifunctional Δ5/Δ6 desaturase was ω-3-specific. Thus, this enzyme(identified herein as SEQ ID NO:18), or one with similar substratespecificity, is desirable upon expression of either the ω-3 Δ6desaturase/Δ6 elongase pathway or the ω-3 Δ9 elongase/Δ8 desaturasepathway for increased synthesis of ω-3 PUFAs.

Of course, in alternate embodiments of the present invention, other DNAswhich are substantially identical to the desaturases and elongasesencoded by SEQ ID NOs:2, 5, 7, 9, 12, 15, 18, 20, 23, 26, 29, 31, 33,35-37, 39, 41, 43, 45, 46, 48-50, 52, 54, 56, 58, 60, 62, 64-68, 70, 78,84, 87, 95, 98, 101 and 105 also can be used for production of DHA inYarrowia lipolytica. By “substantially identical” is intended an aminoacid sequence or nucleic acid sequence exhibiting in order of increasingpreference at least 80%, 90% or 95% homology to the selectedpolypeptides, or nucleic acid sequences encoding the amino acidsequence. For polypeptides, the length of comparison sequences generallyis at least 16 amino acids, preferably at least 20 amino acids or mostpreferably 35 amino acids. For nucleic acids, the length of comparisonsequences generally is at least 50 nucleotides, preferably at least 60nucleotides, more preferably at least 75 nucleotides, and mostpreferably 110 nucleotides.

Homology typically is measured using sequence analysis software, whereinthe term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1.) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc., Madison, Wis.); and 4.)the FASTA program incorporating the Smith-Waterman algorithm (W. R.Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), MeetingDate 1992, 111-20. Suhai, Sandor, Ed. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized. In general, such computer softwarematches similar sequences by assigning degrees of homology to varioussubstitutions, deletions, and other modifications.

In more preferred embodiments, codon-optimized genes encodingdesaturases and elongases that are substantially identical to thosedescribed in SEQ ID NOs: 2, 9, 12, 20, 23, 26, 70, 78, 84,101 and 105are utilized. Specifically, as is well known to one of skill in the art,the expression of heterologous genes can be increased by increasing thetranslational efficiency of encoded mRNAs by replacement of codons inthe native gene with those for optimal gene expression in the selectedhost microorganism. Thus, it is frequently useful to modify a portion ofthe codons encoding a particular polypeptide that is to be expressed ina foreign host, such that the modified polypeptide uses codons that arepreferred by the alternate host; and, use of host preferred codons cansubstantially enhance the expression of the foreign gene encoding thepolypeptide.

In general, host preferred codons can be determined within a particularhost species of interest by examining codon usage in proteins(preferably those expressed in the largest amount) and determining whichare used with highest frequency. Then, the coding sequence for apolypeptide of interest (e.g., a desaturase, elongase, acyltransferase)can be synthesized in whole or in part using the codons preferred in thehost species. All (or portions) of the DNA also can be synthesized toremove any destabilizing sequences or regions of secondary structurethat would be present in the transcribed mRNA. And, all (or portions) ofthe DNA also can be synthesized to alter the base composition to onemore preferable in the desired host cell.

Additionally, the nucleotide sequences surrounding the translationalinitiation codon ‘ATG’ have been found to affect expression in yeastcells. If the desired polypeptide is poorly expressed in yeast, thenucleotide sequences of exogenous genes can be modified to include anefficient yeast translation initiation sequence to obtain optimal geneexpression. For expression in yeast, this can be done by site-directedmutagenesis of an inefficiently expressed gene by fusing it in-frame toan endogenous yeast gene, preferably a highly expressed gene.Alternatively, as demonstrated herein for Yarrowia lipolytica, one candetermine the consensus translation initiation sequence in the host andengineer this sequence into heterologous genes for their optimalexpression in the host of interest.

In the present invention, several desaturase and elongase genes fromTable 5 were codon-optimized for expression in Yarrowia lipolytica,based on the host preferences described above. This was possible byfirst determining the Y. lipolytica codon usage profile (see WO04/101757) and identifying those codons that were preferred. Then, forfurther optimization of gene expression in Y. lipolytica, the consensussequence around the ‘ATG’ initiation codon was determined (i.e.,‘MAMMATGNHS’ (SEQ ID NO:155), wherein the nucleic acid degeneracy codeused is as follows: M=A/C; S=C/G; H=A/C/T; and N=A/C/G/T). Table 7,below, compares the activity of native and codon-optimized genes whenexpressed in Y. lipolytica and provides details about eachcodon-optimized gene. % Sub. Conv. is the abbeviation for “percentsubstrate conversion” and Codon-Opt. is an abbreviation for“codon-optimized”.

TABLE 7 Most Preferred Codon-Optimized Desaturases And Elongases For DHABiosynthesis In Yarrowia lipolytica Codon- Total Bases Opt. Codon-Native Modified In Gene % Opt. Gene % Codon-Opt. Sub. SEQ Native GeneSub. Conv. Gene Conv. Reference ID NO M. alpina Δ6 30% 152 of 1374 bp42% WO 3 desaturase (GenBank (corresponding 04/101753 Accession No. to144 codons) AF465281) M. alpina high affinity 30% 94 of 957 bp 47% WO 24C_(18/20) elongase (corresponding 04/101753 (GenBank Accession to 85codons) No. AX464731) T. aureum C_(18/20) 33% 114 of 817 bp 46% — 27elongase (“ELO2”) (corresponding to 108 codons) S. diclina Δ17 23% 127of 1077 bp 45% Copending 19 desaturase (US (corresponding U.S. Patent2003/0196217 A1) to 117 codons) Application No. 10/840478 Isochrysisgalbana Δ9 — 126 of 789 bp 30% — 71 elongase (corresponding to 123codons) Euglena gracillis Δ8 — 207 of 1263 bp 75% Copending 81desaturase (corresponding U.S. Patent to 192 codons) Application No.11/166993 Isochrysis galbana  7% 203 of 1323 bp 32% — 10 Δ5 desaturase(corresponding to 193 codons) Homo sapiens Δ5 — 227 of 1335 bp 30% — 13desaturase (GenBank (corresponding Accession No. to 207 codons)NP_037534) Thraustochytrium — 170 of 1545 bp 20% — 106 aureum Δ4(corresponding desaturase to 166 codons) Ostreococcus tauri — 160 of 903bp 67% — 102 C_(20/22) elongase (corresponding to 147 codons) Rattusnorvegicus — 127 of 792 bp 43% — 85 C_(16/18) elongase (corresponding(GenBank Accession to 125 codons) No. AB071986)

In additional alternate embodiments of the invention, other DNAs which,although not substantially identical to the preferred desaturases andelongases presented as SEQ ID NOs:3, 10, 13, 19, 24, 27, 71, 81, 85, 102and 106 also can be used for the purposes herein. For example, DNAsequences encoding Δ6 desaturase polypeptides that would be useful forintroduction into Yarrowia lipolytica according to the teachings of thepresent invention may be obtained from microorganisms having an abilityto produce GLA or STA. Such microorganisms include, for example, thosebelonging to the genera Mortierella, Conidiobolus, Pythium,Phytophathora, Penicillium, Porphyridium, Coidosporium, Mucor, Fusarium,Aspergillus, Rhodotorula and Entomophthora. Within the genusPorphyridium, of particular interest is P. cruentum. Within the genusMortierella, of particular interest are M. elongata, M. exigua, M.hygrophila, M. ramanniana var. angulispora and M. alpina. Within thegenus Mucor, of particular interest are M. circinelloides and M.javanicus.

Alternatively, a related desaturase that is not substantially identicalto the M. alpina Δ6 desaturase, for example, but which can desaturate afatty acid molecule at carbon 6 from the carboxyl end of the moleculewould also be useful in the present invention as a Δ6 desaturase,assuming the desaturase can still effectively convert LA to GLA and/orALA to STA. As such, related desaturases and elongases can be identified(or created) by their ability to function substantially the same as thedesaturases and elongases disclosed herein.

As suggested above, in another embodiment one skilled in the art couldcreate a fusion protein having e.g., both Δ12 desaturase and Δ6desaturase activities suitable for the purposes herein. This would bepossible by fusing together a Δ12 desaturase and Δ6 desaturase with anadjoining linker. Either the Δ12 desaturase or the Δ6 desaturase couldbe at the N-terminal portion of the fusion protein. Means to design andsynthesize an appropriate linker molecule are readily known by one ofskill in the art; for example, the linker can be a stretch of alanine orlysine amino acids and will not affect the fusion enzyme's activity.

Finally, it is well known in the art that methods for synthesizingsequences and bringing sequences together are well established in theliterature. Thus, in vitro mutagenesis and selection, site-directedmutagenesis, chemical mutagenesis, “gene shuffling” methods or othermeans can be employed to obtain mutations of naturally occurringdesaturase and/or elongase genes. This would permit production of apolypeptide having desaturase or elongase activity, respectively, invivo with more desirable physical and kinetic parameters for functioningin the host cell (e.g., a longer half-life or a higher rate ofproduction of a desired PUFA).

In summary, although sequences of preferred desaturase and elongasegenes are presented that encode PUFA biosynthetic pathway enzymessuitable for DHA production in Yarrowia lipolytica, these genes are notintended to be limiting to the invention herein. Numerous other genesencoding PUFA biosynthetic pathway enzymes that would be suitable forthe purposes herein could be isolated from a variety of sources (e.g., awildtype, codon-optimized, synthetic and/or mutant enzyme havingappropriate desaturase or elongase activity). These alternatedesaturases would be characterized by the ability to: 1.) desaturate afatty acid between the 17^(th) and 18^(th) carbon atom numbered from thecarboxyl-terminal end of the molecule and catalyze the conversion of ARAto EPA and DGLA to ETA (Δ17 desaturases); 2.) catalyze the conversion ofLA to GLA and/or ALA to STA (Δ6 desaturases); 3.) catalyze theconversion of DGLA to ARA and/or ETA to EPA (Δ5 desaturases); 4.)catalyze the conversion of oleic acid to LA (Δ12 desaturases); 5.)catalyze the conversion of LA to ALA (Δ15 desaturases); 6.) catalyze theconversion of EDA to DGLA and/or ETrA to ETA (Δ8 desaturases); 7.)catalyze the conversion of DPA to DHA (Δ4 desaturases); and/or 8.)catalyze the conversion of palmitate to palmitoleic acid and/or stearateto oleic acid (Δ9 desaturases). In like manner, suitable elongases forthe purposes herein are not limited to those from a specific source;instead, the enzymes having use for the purposes herein arecharacterized by their ability to elongate a fatty acid carbon chain by2 carbons relative to the substrate the elongase acts upon, to therebyproduce a mono- or polyunsaturated fatty acid. More specifically, theseelongases would be characterized by the ability to: 1.) elongate LA toEDA and/or ALA to ETrA (Δ9 elongases); 2.) elongate a C18 substrate toproduce a C20 product (C_(18/20)elongases); 3.) elongate a C14 substrateto produce a C16 product (C_(14/16)elongases); 4.) elongate a C16substrate to produce a C18 product (C_(16/18)elongases); and/or 5.)elongate a C20 substrate to produce a C22 product (C_(20/22)elongases).Again, it is important to note that some elongases may be capable ofcatalyzing several elongase reactions, as a result of broad substratespecificity.

Acyltransferases and Their Role in the Terminal Step of TAG Biosynthesis

Acyltransferases are intimately involved in the biosynthesis of TAGs.Two comprehensive mini-reviews on TAG biosynthesis in yeast, includingdetails concerning the genes involved and the metabolic intermediatesthat lead to TAG synthesis are: D. Sorger and G. Daum, Appl. Microbiol.Biotechnol. 61:289-299 (2003); and H. Müllner and G. Daum, ActaBiochimica Polonica, 51(2):323-347 (2004). Although the authors of thesereviews clearly summarize the different classes of eukaryoticacyltransferase gene families (infra), they also acknowledge thatregulatory aspects of TAG synthesis and formation of neutral lipids inlipid particles remain far from clear.

Four eukaryotic acyltransferase gene families have been identified whichare involved in acyl-CoA-dependent or independent esterificationreactions leading to neutral lipid synthesis:

-   -   (1) The acyl-CoA:cholesterol acyltransferase (ACAT) family, EC        2.3.1.26 (commonly known as sterol acyltransferases). This        family of genes includes enzymes responsible for the conversion        of acyl-CoA and sterol to CoA and sterol esters. This family        also includes DGAT1, involved in the terminal step of TAG        biosynthesis.    -   (2) The lecithin:cholesterol acyltransferase (LCAT) family, EC        2.3.1.43. This family of genes is responsible for the conversion        of phosphatidylcholine and a sterol to a sterol ester and        1-acylglycerophosphocholine. This family also includes the        phospholipid:diacylglycerol acyltransferase (PDAT) enzyme        involved in the transfer of an acyl group from the sn-2 position        of a phospholipid to the sn-3 position of 1,2-diacylglycerol        resulting in TAG biosynthesis.    -   (3) The diacylglycerol acyltransferase (DAG AT) family, EC        2.3.1.20. This family of genes (which includes DGAT2) is        involved in the terminal step of TAG biosynthesis.    -   (4) The glycerol-3-phosphate acyltransferase and acyl-COA        lysophosphatidic acid acyltransferase (GPAT/LPAAT) family. GPAT        (E.C. 2.3.1.15) proteins are responsible for the first step of        TAG biosynthesis, while LPAAT (E.C. 2.3.1.51) enzymes are        involved in the second step of TAG biosynthesis. This family        also includes lysophosphatidylcholine acyltransferase (LPCAT)        that catalyzes the acyl exchange between phospholipid and CoA.        Together, these 4 acyltransferase gene families represent        overlapping biosynthetic systems for neutral lipid formation and        appear to be the result of differential regulation, alternate        localization, and different substrate specifities (H. Müllner        and G. Daum, supra). Each of these four gene families will be        discussed herein based on their importance with respect to        metabolic engineering in Yarrowia lipolytica, to enable        synthesis of greater than 5% DHA.

The Functionality Of Various Acyltransferases

The interplay between many of these acyltransferases in Yarrowialipolytica is schematically diagrammed in FIG. 4. Focusing initially onthe direct mechanism of TAG biosynthesis, the first step in this processis the esterification of one molecule of acyl-CoA tosn-glycerol-3-phosphate via GPAT to produce lysophosphatidic acid (LPA)(and CoA as a by-product). Then, lysophosphatidic acid is converted tophosphatidic acid (PA) (and CoA as a by-product) by the esterificationof a second molecule of acyl-CoA, a reaction that is catalyzed by LPAAT.Phosphatidic acid phosphatase is then responsible for the removal of aphosphate group from phosphatidic acid to yield 1,2-diacylglycerol(DAG). And, finally a third fatty acid is added to the sn-3 position ofDAG by a DAG AT (e.g., DGAT1, DGAT2 or PDAT) to form TAG.

Historically, DGAT1 was thought to be the only enzyme specificallyinvolved in TAG synthesis, catalyzing the reaction responsible for theconversion of acyl-CoA and DAG to TAG and CoA, wherein an acyl-CoA groupis transferred to. DAG to form TAG. DGAT1 was known to be homologous toACATs; however, recent studies have identified a new family of DAG ATenzymes that are unrelated to the ACAT gene family. Thus, nomenclaturenow distinguishes between the DAG AT enzymes that are related to theACAT gene family (DGAT1 family) versus those that are unrelated (DGAT2family) (Lardizabal et al., J. Biol. Chem. 276(42):38862-38869 (2001)).Members of the DGAT2 family have been identified in all major phyla ofeukaryotes (fungi, plants, animals and basal eukaryotes).

Even more recently, Dahlqvist et al. (Proc. Nat. Acad. Sci. (USA)97:6487-6492 (2000)) and Oelkers et al. (J. Biol. Chem. 275:15609-15612(2000)) discovered that TAG synthesis can also occur in the absence ofacyl-CoA, via an acyl-CoA-independent mechanism. Specifically, PDATremoves an acyl group from the sn-2 position of a phosphotidylcholinesubstrate for transfer to DAG to produce TAG. This enzyme isstructurally related to the LCAT family; and although the function ofPDAT is not as well characterized as DGAT2, PDAT has been postulated toplay a major role in removing “unusual” fatty acids from phospholipidsin some oilseed plants (Banas, A. et al., Biochem. Soc. Trans.28(6):703-705 (2000)).

With respect to TAG synthesis in Saccharomyces cerevisiae, threepathways have been described (Sandager, L. et al., J. Biol. Chem.277(8):6478-6482 (2002)). First, TAGs are mainly synthesized from DAGand acyl-CoAs by the activity of DGAT2 (encoded by the DGA1 gene). Morerecently, however, a PDAT (encoded by the LRO1 gene) has also beenidentified. Finally, two acyl-CoA:sterol-acyltransferases (encoded bythe ARE1 and ARE2 genes) are known that utilize acyl-CoAs and sterols toproduce sterol esters (and TAGs in low quantities; see Sandager et al.,Biochem. Soc. Trans. 28(6):700-702 (2000)). Together, PDAT and DGAT2 areresponsible for approximately 95% of oil biosynthesis in S. cerevisiae.

Based on several publicly available sequences encoding DGAT1s, DGAT2s,PDATs and ARE2s (infra), the Applicants isolated and characterized thegenes encoding DGAT1 (SEQ ID NO:122), DGAT2 (SEQ ID NOs:130, 132 and 134[wherein SEQ ID NO:130 contains at least two additional nested ORFs asprovided in SEQ ID NOs:132 and 134; the ORF encoded by SEQ ID NO:1 34has a high degree of similarity to other known DGAT enzymes anddisruption in SEQ ID NO:134 eliminated DGAT function of the native gene,thereby confirming that the polypeptide of SEQ ID NO:135 has DGATfunctionality]), PDAT (SEQ ID NO:117) and ARE2 (SEQ ID NO:119) inYarrowia lipolytica. In contrast to the model developed in S.cerevisiae, wherein PDAT and DGAT2 are responsible for approximately 95%of oil biosynthesis, however, it was discovered that the PDAT, DGAT2 andDGAT1 of Yarrowia lipolytica are responsible for up to ˜95% of oilbiosynthesis (while ARE2 may additionally be a minor contributor to oilbiosynthesis).

The final acyltransferase enzyme whose function could be important inthe accumulation of DHA in the TAG fraction of Yarrowia lipolytica isLPCAT. As shown in FIG. 4, this enzyme (EC 2.3.1.23) is hypothesized tobe responsible for two-way acyl exchange at the sn-2 position ofsn-phosphatidylcholine to enhance ω-6 and ω-3 PUFA biosynthesis. Thishypothesis is based on the following studies: (1) Stymne S. and A. K.Stobart (Biochem J. 223(2):305-14(1984)), who hypothesized that LPCATaffected exchange between the acyl-CoA pool and phosphatidylcholine (PC)pool; (2) Domergue, F. et al. (J. Bio. Chem 278:35115 (2003)), whosuggested that accumulation of GLA at the sn-2 position of PC and theinability to efficiently synthesize ARA in yeast was a result of theelongation step involved in PUFA biosynthesis occurring within theacyl-CoA pool, while Δ5 and Δ6 desaturation steps occurred predominantlyat the sn-2 position of PC; (3) Abbadi, A. et al. (The Plant Cell,16:2734-2748 (2004)), who suggested that LPCAT plays a criticial role inthe successful reconstitution of a Δ6 desaturase/Δ6 elongase pathway,based on analysis on the constraints of PUFA accumulation in transgenicoilseed plants; and, (4) WO 2004/076617 Δ2 (Renz, A. et al.), whoprovided a gene encoding LPCAT from Caenorhabditis elegans (T06E8.1)that substantially improved the efficiency of elongation in agenetically introduced Δ6 desaturase/Δ6 elongase pathway in S.cerevisiae. The inventors concluded that LPCAT allowed efficient andcontinuous exchange of the newly synthesized fatty acids betweenphospholipids and the acyl-CoA pool, since desaturases catalyze theintroduction of double bonds in lipid-coupled fatty acids (sn-2 acyl PC)while elongases exclusively catalyze the elongation of CoA esterifiedfatty acids (acyl-CoAs).

Selection of Heterologous Acyltransferase Genes for DHA Synthesis

Since naturally produced PUFAs in Yarrowia lipolytica are limited to18:2 fatty acids (and less commonly, 18:3 fatty acids), it would belikely that the host organism's native genes encoding GPAT; LPAAT (i.e.,LPMT1 or LPAAT2), DGAT1, DGAT2, PDAT and LPCAT could have difficultyefficiently synthesizing TAGs comprising fatty acids that were 18:3 andgreater in length (e.g., DHA). Thus, in some cases, a heterologous (or“foreign”) acyltransferase could be preferred over a native enzyme.

Numerous acyltransferase genes have been identified in various organismsand disclosed in the public and patent literature. For instance, thefollowing GenBank Accession Numbers refer to examples of publiclyavailable acyltransferase genes useful in lipid biosynthesis: CQ891256,AY441057, AY360170, AY318749, AY093169, AJ422054, AJ311354, AF251795,Y00771, M77003 (GPATs); Q93841, Q22267, Q99943, O15120, Q9NRZ7, Q9NRZ5,Q9NUQ2, O35083, Q9D1E8, Q924S1, Q59188, Q42670, P26647, P44848, Q9ZJN8,O25903 Q42868, Q42870, P26974, P33333, Q9XFW4, CQ891252, CQ891250,CQ891260, CQ891.258, CQ891248, CQ891245, CQ891241, CQ891238, CQ891254,CQ891235 (LPAATs); AY445635, BC003717, NM_(—)010046, NM_(—)053437,NM_(—)174693, AY116586, AY327327, AY327326, AF298815 and AF164434(DGAT1s); and NC_(—)001147 [locus NP_(—)014888], NM_(—)012079,NM_(—)127503, AF051849, AJ238008, NM_(—)026384, NM_(—)010046, AB057816,AY093657, AB062762, AF221132, AF391089, AF391090, AF129003, AF251794 andAF164434 (DGAT2s); P40345, O94680, NP_(—)596330, NP_(—)190069 andAB006704 [gi:2351069] (PDATs). Similarly, the patent literature providesmany additional DNA sequences of genes (and/or details concerningseveral of the genes above and their methods of isolation) involved inTAG production [e.g., U.S. Pat. No. 5,210,189, WO 2003/025165 (GPATs);EP1144649 A2, EP1131438, U.S. Pat. No. 5,968,791, U.S. Pat. No.6,093,568, WO 2000/049156 and WO 2004/087902 (LPAATs); U.S. Pat. No.6,100,077, U.S. Pat. No. 6,552,250, U.S. Pat. No. 6,344,548, US2004/0088759A1 and US 20040078836A1 (DGAT1s); US 2003/124126, WO2001/034814, US2003/115632, US2003/0028923 and US 2004/0107459 (DGAT2s);WO 2000/060095 (PDATs); and WO 2004/076617 A2 (LPCATs).

The examples above are not intended to be limiting and numerous othergenes encoding DGAT1, DGAT2, PDAT, GPAT, LPCAT and LPAAT derived fromdifferent sources would be suitable for introduction into Yarrowialipolytica. For example, the Applicants have identified novel DGAT1sfrom Mortierella alpina (SEQ ID NOs:124 and 125), Neurospora crassa (SEQID NO:126), Gibberella zeae PH-1 (SEQ ID NO:127), Magnaporthe grisea(SEQ ID NO:128) and Aspergillus nidulans (SEQ ID NO:129); and, a novelDGAT2 (SEQ ID NOs:136 and 137), GPAT (SEQ ID NOs:138 and 139), LPAAT1(SEQ ID NOs:108 and 109) and LPAAT2 (SEQ ID NOs:110 and 111) fromMortierella alpina.

Preferred Acyltransferase Genes For DHA Svnthesis

Despite the wide selection of acyltransferases that could be suitablefor expression in Yarrowia lipolytica, however, in preferred embodimentsof the present invention the DGAT1, DGAT2, PDAT, GPAT, LPAAT and LPCATare selected from organisms producing significant amounts of longerchain ω-6 (e.g., ARA) and/or ω-3 (e.g., EPA, DHA) PUFAs. Thus, thefollowing enzymes are especially preferred (or derivatives thereof):

TABLE 8 Preferred Heterologous Acyltransferases For Expression In A HighDHA-Producing Strain Of Yarrowia lipolytica SEQ ID ORF OrganismReference NOs DGAT1 Mortierella Co-pending U.S. Patent 124, 125 alpinaApplication Number 11/024544 DGAT2 Mortierella Co-pending U.S. Patent136, 137 alpina Application Number 11/024545 GPAT Mortierella — 138, 139alpina LPAAT1 Mortierella — 108, 109 alpina LPAAT2 MortierellaCo-pending U.S. Patent 110, 111 alpina Application Number 60/689031LPCAT Caenorhabditis Clone T06E8.1; 121 elegans WO 2004/076617 A2

Although not intended to be limiting in the invention herein, M. alpinawas selected as a preferred source of heterologous acyltransferasessince the native organism is capable of synthesizing ARA atconcentrations greater than 50% of the total fatty acids (TFAs). Insimilar manner, C. elegans can produce up to 20-30% of its TFAs as EPA.

Of course, in alternate embodiments of the present invention, other DNAswhich are substantially identical to the acyltransferases encoded by SEQID NOs:108-111, 121, 124, 125 and 136-139 also can be used forheterologous expression in Yarrowia lipolytica to facilitate theproduction and accumulation of DHA in the TAG fraction. In morepreferred embodiments, codon-optimized genes encoding acyltransferasesthat are substantially identical to those described in SEQ ID NOs:108-111, 121, 124, 125 and 136-139 are utilized.

General Expression Systems, Cassettes, Vectors and Transformation forExpression of Foreign Genes

Microbial expression systems and expression vectors containingregulatory sequences that direct high-level expression of foreignproteins such as those leading to the high-level production of DHA arewell known to those skilled in the art. Any of these could be used toconstruct chimeric genes encoding the preferred desaturases, elongasesand acyltransferases. These chimeric genes could then be introduced intoYarrowia lipolytica using standard methods of transformation to providehigh-level expression of the encoded enzymes.

Vectors or DNA cassettes useful for the transformation of host cells arewell known in the art. The specific choice of sequences present in theconstruct is dependent upon the desired expression products, the natureof the host cell, and the proposed means of separating transformed cellsversus non-transformed cells. Typically, however, the vector or cassettecontains sequences directing transcription and translation of therelevant gene(s), a selectable marker and sequences allowing autonomousreplication or chromosomal integration. Suitable vectors comprise aregion 5′ of the gene that controls transcriptional initiation (e.g., apromoter) and a region 3′ of the DNA fragment that controlstranscriptional termination (i.e., a terminator). It is most preferredwhen both control regions are derived from genes from the transformedhost cell, although it is to be understood that such control regionsneed not be derived from the genes native to the specific species chosenas a production host.

Where two or more genes are expressed from separate replicating vectors,it is desirable that each vector has a different means of selection andshould lack homology to the other constructs to maintain stableexpression and prevent reassortment of elements among constructs.Judicious choice of regulatory regions, selection means and method ofpropagation of the introduced construct can be experimentally determinedso that all introduced genes are expressed at the necessary levels toprovide for synthesis of the desired products.

Constructs comprising the gene(s) of interest may be introduced into ahost cell by any standard technique. These techniques includetransformation (e.g., lithium acetate transformation [Methods inEnzymology, 194:186-187 (1991)]), protoplast fusion, bolistic impact,electroporation, microinjection, or any other method that introduces thegene(s) of interest into the host cell. More specific teachingsapplicable for Yarrowia lipolytica include U.S. Pat. No. 4,880,741 andU.S. Pat. No. 5,071,764 and Chen, D. C. et al. (Appl MicrobiolBiotechnol. 48(2):232.-235 (1997)).

For convenience, a host cell that has been manipulated by any method totake up a DNA sequence (e.g., an expression cassette) will be referredto as “transformed” or “recombinant” herein. The transformed host willhave at least one copy of the expression construct and may have two ormore, depending upon whether the gene is integrated into the genome,amplified, or is present on an extrachromosomal element having multiplecopy numbers. The transformed host cell can be identified by variousselection techniques, as described in WO 2004/101757 and WO2005/003310.

Preferred selection methods for use herein are resistance to kanamycin,hygromycin and the amino glycoside G418, as well as ability to grow onmedia lacking uracil, leucine or histidine. In alternate embodiments,5-fluoroorotic acid (5-fluorouracil-6-carboxylic acid monohydrate;“5-FOA”) is used for selection of yeast Ura⁻ mutants. The compound istoxic to yeast cells that possess a functioning URA3 gene encodingorotidine 5′-monophosphate decarboxylase (OMP decarboxylase); thus,based on this toxicity, 5-FOA is especially useful for the selection andidentification of Ura⁻ mutant yeast strains (Bartel, P. L. and Fields,S., Yeast 2-Hybrid System, Oxford University: New York, v. 7, pp109-147, 1997).

An alternate preferred selection method utilized herein relies on adominant, non antibiotic marker for Yarrowia lipolytica based onsulfonylurea resistance. The technique is also generally applicable toother industrial yeast strains that may be haploid, diploid, aneuploidor heterozygous. It is expected to overcome two main limitations to thedevelopment of genetic transformation systems for industrial yeaststrains, wherein: (1) there are almost no naturally auxotrophic strains,and the isolation of spontaneous or induced auxotrophic mutants ishindered by the ploidy of the strains; and, (2) the use of antibioticresistance markers may limit the commercial application of strains dueto restrictions on the release of genetically modified organismscarrying antibiotic resistance genes. Although Puig et al. (J. Agric.Food Chem. 46:1689-1693 (1998)) developed a method to overcome theselimitations based on the genetic engineering of a target strain in orderto make it auxotrophic for uridine and the subsequent use of the URA3marker in order to introduce traits of interest, this strategy wasdeemed too laborious for routine work.

The new sulfonylurea resistance selection marker disclosed herein fortransforming Yarrowia lipolytica does not rely on a foreign gene but ona mutant native gene. Thus, it neither requires auxotrophy nor resultsin auxotrophy and allows transformation of wild type strains. Morespecifically, the marker gene (SEQ ID NO:243) is a nativeacetohydroxyacid synthase (AHAS or acetolactate synthase; E. C.4.1.3.18) that has a single amino acid change (W497L) that conferssulfonyl urea herbicide resistance. AHAS is the first common enzyme inthe pathway for the biosynthesis of branched-chain amino acids and it isthe target of the sulfonylurea and imidazolinone herbicides. The W497Lmutation is known, based on work in Saccharomyces cerevisiae (Falco, S.C., et al., Dev. Ind. Microbiol. 30:187-194 (1989); Duggleby, R. G., et.al. Eur. J. Biochem. 270:2895 (2003). Initial testing determined thatYarrowia cells were not naturally resistant to the herbicide as a resultof: 1.) poor or no uptake of the herbicide; 2.) the presence of a nativeherbicide-resistant form of AHAS; and/or 3.) use of aherbicide-inactivating mechanism. This thereby enabled synthesis and useof the mutant AHAS gene (SEQ ID NO:243) as a means for selection oftransformants.

An additional method for recyling a selection marker relies onsite-specific recombinase systems. Briefly, the site-specificrecombination system consists of two elements: (1) a recombination sitehaving a characteristic DNA sequence [e.g., LoxP]; and (2) a recombinaseenzyme that binds to the DNA sequence specifically and catalyzesrecombination (i.e., excision) between DNA sequences when two or more ofthe recombination sites are oriented in the same direction at a giveninterval on the same DNA molecule [e.g., Cre]. This methodology hasutility as a means of selection, since it is possible to “recycle” apair of preferred selection markers for their use in multiple sequentialtransformations.

Specifically, an integration construct is created comprising a targetgene that is desirable to insert into the host genome (e.g., adesaturase, elongase, acyltransferase), as well as a first selectionmarker (e.g., Ura3, hygromycin phosphotransferase [HPT]) that is flankedby recombination sites. Following transformation and selection of thetransformants, the first selection marker is excised from the chromosomeby the introduction of a replicating plasmid carrying a second selectionmarker (e.g., sulfonylurea resistance [AHAS]) and a recombinase suitableto recognize the site-specific recombination sites introduced into thegenome. Upon selection of those transformants carrying the second markerand confirmation of excision of the first selection marker from the hostgenome, the replicating plasmid is then cured from the host in theabsence of selection. This produces a transformant that possessesneither the first nor second selection marker, and thus the cured strainis available for another round of transformation. One skilled in the artwill recognize that the methodology is not limited to the particularselection markers or site-specific recombination system used in thepresent invention.

Overexpression of Foreign Genes in Yarrowia lipolytica

As is well known to one of skill in the art, merely inserting a gene(e.g., a desaturase) into a cloning vector does not ensure that it willbe successfully expressed at the level needed. It may be desirable tomanipulate a number of different genetic elements that control aspectsof transcription, translation, protein stability, oxygen limitation andsecretion from the host cell. More specifically, gene expression may becontrolled by altering the following: the nature of the relevanttranscriptional promoter and terminator sequences; the number of copiesof the cloned gene; whether the gene is plasmid-borne or integrated intothe genome of the host cell; the final cellular location of thesynthesized foreign protein; the efficiency of translation in the hostorganism; the intrinsic stability of the cloned gene protein within thehost cell; and the codon usage within the cloned gene, such that itsfrequency approaches the frequency of preferred codon usage of the hostcell. Several of these methods of overexpression will be discussedbelow, and are useful in the present invention as a means to overexpresse.g., desaturases, elongases and acyltransferases in Yarrowialipolytica.

Expression of the desired gene(s) can be increased at thetranscriptional level through the use of a stronger promoter (eitherregulated or constitutive) to cause increased expression, byremoving/deleting destabilizing sequences from either the mRNA or theencoded protein, or by adding stabilizing sequences to the mRNA (U.S.pat. No. 4,910,141).

Initiation control regions or promoters which are useful to driveexpression of desaturase, elongase and acyltransferase genes in thedesired host cell are numerous and familiar to those skilled in the art.Virtually any promoter capable of directing expression of these genes inYarrowia lipolytica is suitable for the present invention. Expression inthe host cell can be accomplished in a transient or stable fashion.Transient expression can be accomplished by inducing the activity of aregulatable promoter operably linked to the gene of interest;alternatively, stable expression can be achieved by the use of aconstitutive promoter operably linked to the gene of interest. As anexample, when the host cell is yeast, transcriptional and translationalregions functional in yeast cells are provided, particularly from thehost species. The transcriptional initiation regulatory regions can beobtained, for example, from: 1.) genes in the glycolytic pathway, suchas alcohol dehydrogenase, glyceraldehyde-3-phosphate-dehydrogenase,phosphoglycerate mutase, fructose-bisphosphate aldolase,phosphoglucose-isomerase, phosphoglycerate kinase, glycerol-3-phosphateO-acyltransferase, etc.; or 2.) regulatable genes, such as acidphosphatase, lactase, metallothionein, glucoamylase, the translationelongation factor EF1-α (TEF) protein (U.S. Pat. No. 6,265,185),ribosomal protein S7 (U.S. Pat. No. 6,265,185), ammonium transporterproteins, export proteins, etc. Any one of a number of regulatorysequences can be used, depending upon whether constitutive or inducedtranscription is desired, the efficiency of the promoter inexpressing-the ORF of interest, the ease of construction and the like.The examples provided above are not intended to be limiting in theinvention herein.

As one of skill in the art is aware, a variety of methods are availableto compare the activity of various promoters. This type of comparison isuseful to facilitate a determination of each promoter's strength for usein future applications wherein a suite of promoters would be necessaryto construct chimeric genes useful for the production of ω-6 and ω-3fatty acids. Thus, it may be useful to indirectly quantitate promoteractivity based on reporter gene expression (i.e., the E. coli geneencoding β-glucuronidase (GUS)). In alternate embodiments, it maysometimes be useful to quantify promoter activity using morequantitative means. One suitable method is the use of real-time PCR (fora general review of real-time PCR applications, see Ginzinger, D. J.,Experimental Hematology, 30:503-512 (2002)). Real-time PCR is based onthe detection and quantitation of a fluorescent reporter. This signalincreases in direct proportion to the amount of PCR product in areaction. By recording the amount of fluorescence emission at eachcycle, it is possible to monitor the PCR reaction during exponentialphase where the first significant increase in the amount of PCR productcorrelates to the initial amount of target template. There are twogeneral methods for the quantitative detection of the amplicon: (1) useof fluorescent probes; or (2) use of DNA-binding agents (e.g.,SYBR-green I, ethidium bromide). For relative gene expressioncomparisons, it is necessary to use an endogenous control as an internalreference (e.g., a chromosomally encoded 16S rRNA gene), therebyallowing one to normalize for differences in the amount of total DNAadded to each real-time PCR reaction. Specific methods for real-time PCRare well documented in the art. See, for example, the Real Time PCRSpecial Issue (Methods, 25(4):383-481 (2001)).

Following a real-time PCR reaction, the recorded fluorescence intensityis used to quantitate the amount of template by use of: 1.) an absolutestandard method (wherein a known amount of standard such as in vitrotranslated RNA (cRNA) is used); 2.) a relative standard method (whereinknown amounts of the target nucleic acid are included in the assaydesign in each run); or 3.) a comparative C_(T) method (ΔΔCT) forrelative quantitation of gene expression (wherein the relative amount ofthe target sequence is compared to any of the reference values chosenand the result is given as relative to the reference value). Thecomparative C_(T) method requires one to first determine the difference(ΔC_(T)) between the C_(T) values of the target and the normalizer,wherein: ΔC_(T)=C_(T) (target)−C_(T)(normalizer). This value iscalculated for each sample to be quantitated and one sample must beselected as the reference against which each comparison is made. Thecomparative ΔΔC_(T) calculation involves finding the difference betweeneach sample's ΔC_(T) and the baseline's ΔC_(T), and then transformingthese values into absolute values according to the formula 2^(−ΔΔCT).

Despite the wide selection of promoters that could be suitable forexpression in Yarrowia lipolytica, however, in preferred embodiments ofthe present invention the promoters are selected from those shown belowin Table 9 (or derivatives thereof).

TABLE 9 Native Promoters Preferred For Overexpression In Yarrowialipolytica SEQ Promoter Activity ID Name Location* Native Gene “Rank”Reference NO TEF — translation 1 U.S. Pat. No. 6,265,185 218 elongation(Muller et al.); factor EF1-α GenBank Accession No. AF054508 GPD −968 bpto +3 bp glyceraldehyde- 2 WO 210 3-phosphate- 2005/003310 dehydrogenaseGPM −875 bp to +3 bp phospho- 1 WO 212 glycerate 2005/003310 mutase FBA−1001 bp to fructose- 4 WO 213 −1 bp bisphosphate 2005/049805 aldolaseFBAIN −804 bp to fructose- 7 WO 214 +169 bp bisphosphate 2005/049805(including a aldolase 102 bp intron [+64 to +165]) FBAINm −804 bp tofructose- 5 WO 215 +169 bp with bisphosphate 2005/049805 modification***aldolase GPDIN −973 bp to glyceraldehyde- 3 Co-pending U.S. 211 +201 bp3-phosphate- Patent (including a dehydrogenase Application No. 146 bpintron 11/183664 [+49 to +194]) GPAT −1130 to +3 bp glycerol-3- 5Co-pending U.S. 216 phosphate O- Patent acyltransferase Application No.11/225354 YAT1 −778 to −1 bp ammonium 6 Co-pending U.S. 217 transporterPatent enzyme Application No. 11/185301 EXP1 −1000 to −1 bp exportprotein 6 — 221 *Location is with respect to the native gene, whereinthe ‘A’ position of the ‘ATG’ translation initiation codon is designatedas +1. ***The FBAINm promoter is a modified version of the FBAINpromoter, wherein FBAINm has a 52 bp deletion between the ATGtranslation initiation codon and the intron of the FBAIN promoter(thereby including only 22 amino acids of the N-terminus) and a newtranslation consensus motif after the intron. Furthermore, while theFBAIN promoter generates a fusion protein when fused with the codingregion of a gene to be expressed, the FBAINm promoter does not generatesuch a fusion protein.

The activity of GPM is about the same as TEF, while the activity of GPD,FBA, FBAIN, FBAINm, GPDIN, GPAT, YAT1 and EXP1 are all greater than TEF(activity is quantified in a relative manner in the column titled“Activity Rank”, wherein a ‘1’ corresponds to the promoter with lowestactivity, while a ‘7’ corresponds to the promoter with highestactivity). This quantitation is based on comparative studies whereineach promoter was used for creation of a chimeric gene possessing the E.coli gene encoding β-glucuronidase (GUS) as a reporter (Jefferson, R. A.Nature. 14;342:837-838 (1989)) and a ˜100 bp of the 3′ region of theYarrowia Xpr gene. GUS activity in each expressed construct was measuredby histochemical and/or fluorometric assays (Jefferson, R. A. Plant Mol.Biol. Reporter5:387-405 (1987)) and/or by use of Real Time PCR.

The YAT1 promoter is unique in that it is characterized by theApplicants as the first promoter identified within Yarrowia that isinducible under oleaginous conditions (i.e., nitrogen limitation).Specifically, although the YAT1 promoter is active in media containingnitrogen (e.g., up to about 0.5% ammonium sulfate), the activity of thepromoter increases when the host cell is grown in nitrogen-limitingconditions (e.g., in medium containing very low levels of ammonium, orlacking ammonium). Thus, a preferred medium would be one that containsless than about 0.1% ammonium sulfate, or other suitable ammonium salts.In a more preferred embodiment, the YAT1 promoter is induced when thehost cell is grown in media with a high carbon to nitrogen (i.e., C:N)ratio, such as a high glucose medium (HGM) containing about 8-12%glucose, and about 0.1% or less ammonium sulfate. These conditions arealso sufficient to induce oleaginy in those yeast that are oleaginous(e.g., Yarrowia lipolytica). Based on GUS activity of cell extracts, theactivity of the YAT1 promoter increased by ˜37 fold when cells wereswitched from a minimal medium into HGM and grown for 24 hrs; after 120hrs in HGM, the activity was reduced somewhat but was still 25× higherthan the activity in minimal medium comprising nitrogen (Example 1).

Of course, in alternate embodiments of the present invention, otherpromoters which are derived from any of the promoter regions describedabove in Table 9 also can be used for heterologous expression inYarrowia lipolytica to facilitate the production and accumulation of DHAin the TAG fraction. In particular, modification of the lengths of anyof the promoters described above can result in a mutant promoter havingidentical activity, since the exact boundaries of these regulatorysequences have not been completely defined. In alternate embodiments,the enhancers located within the introns of the FBAIN and GPDINpromoters can be used to create a chimeric promoter having increasedactivity relative to the native Yarrowia promoter (e.g., chimericGPM::FBAIN and GPM::GPDIN promoters (SEQ ID NOs:219 and 220) hadincreased activity relative to the GPM promoter alone, when drivingexpression of the GUS reporter gene in conjunction with a ˜100 bp of the3′ region of the Yarrowia Xpr gene).

The termination region can be derived from the 3′ region of the genefrom which the initiation region was obtained or from a different gene.A large number of termination regions are known and functionsatisfactorily in a variety of hosts (when utilized both in the same and30 different genera and species from where they were derived). Thetermination region usually is selected more as a matter of conveniencerather than because of any particular property. Preferably, thetermination region is derived from a yeast gene, particularlySaccharomyces, Schizosaccharomyces, Candida, Yarrowia or Kluyveromyces.The 3′-regions of mammalian genes encoding γ-interferon and α-2interferon are also known to function in yeast. Termination controlregions may also be derived from various genes native to the preferredhosts. Optionally, a termination site may be unnecessary; however, it ismost preferred if included. Although not intended to be limiting,termination regions useful in the disclosure herein include: ˜100 bp ofthe 3′ region of the Yarrowia lipolytica extracellular protease (XPR;GenBank Accession No. M17741); the acyl-coA oxidase (Aco3: GenBankAccession No. AJ001301 and No. CAA04661; Pox3: GenBank Accession No.XP_(—)503244) terminators; the Pex20 (GenBank Accession No. AF054613)terminator; the Pex16 (GenBank Accession No. U75433) terminator; theLip1 (GenBank Accession No. Z50020) terminator; the Lip2 (GenBankAccession No. AJ012632) terminator: and the 3-oxoacyl-coA thiolase (OCT;GenBank Accession No. X69988) terminator.

Additional copies (i.e., more than one copy) of the desaturase, elongaseand/or acyltransferase genes described above may be introduced intoYarrowia lipolytica to thereby increase DHA production and accumulation.Specifically, additional copies of genes may be cloned within a singleexpression construct; and/or, additional copies of the cloned gene(s)may be introduced into the host cell by increasing the plasmid copynumber or by multiple integration of the cloned gene into the genome(infra). For example, in one embodiment, a strain of Yarrowia lipolytica(i.e., strain Y3000) was engineered to produce greater than 5% DHA bythe introduction and integration into the Yarrowia genome of chimericgenes comprising: 3 copies of a Δ12 desaturase, 2 copies of a Δ6desaturase, 4 copies of a C_(18/20)elongase, 5 copies of a Δ5desaturase, 3 copies of a Δ17 desaturase, 3 copies of aC_(16/18)elongase, 1 copy of a C_(20/22)elongase and 1 copy of a Δ4desaturase.

In general, once the DNA that is suitable for expression in anoleaginous yeast has been obtained (e.g., a chimeric gene comprising apromoter, ORF and terminator), it is placed in a plasmid vector capableof autonomous replication in a host cell; or, it is directly integratedinto the genome of the host cell. Integration of expression cassettescan occur randomly within the host genome or can be targeted through theuse of constructs containing regions of homology with the host genomesufficient to target recombination with the host locus. Although notrelied on in the present invention, all or some of the transcriptionaland translational regulatory regions can be provided by the endogenouslocus where constructs are targeted to an endogenous locus.

In the present invention, the preferred method of expressing genes inYarrowia lipolytica is by integration of linear DNA into the genome ofthe host; and, integration into multiple locations within the genome canbe particularly useful when high level expression of genes are desired.Toward this end, it is desirable to identify a sequence within thegenome that is present in multiple copies.

Schmid-Berger et al. (J. Bact. 176(9):2477-2482 (1994)) discovered thefirst retrotransposon-like element Ylt1 in Yarrowia lipolytica. Thisretrotransposon is characterized by the presence of long terminalrepeats (LTRs; each approximately 700 bp in length) called zeta regions.Ylt1 and solo zeta elements were present in a dispersed manner withinthe genome in at least 35 copies/genome and 50-60 copies/genome,respectively; both elements were determined to function as sites ofhomologous recombination. Further, work by Juretzek et al. (Yeast18:97-113 (2001)) demonstrated that gene expression could bedramatically increased by targeting plasmids into the repetitive regionsof the yeast genome (using linear DNA with LTR zeta regions at bothends), as compared to the expression obtained using low-copy plasmidtransformants. Thus, zeta-directed integration can be ideal as a meansto ensure multiple integration of plasmid DNA into Y. lipolytica,thereby permitting high-level gene expression. Unfortunately, however,not all strains of Y. lipolytica possess zeta regions (e.g., the strainidentified as ATCC #20362). When the strain lacks such regions, it isalso possible to integrate plasmid DNA comprising expression cassettesinto alternate loci to reach the desired copy number for the expressioncassette. For example, preferred alternate loci include: the Ura3 locus(GenBank Accession No. AJ306421), the Leu2 gene locus (GenBank AccessionNo. AF260230), the Lys5 gene (GenBank Accession No. M34929), the Aco2gene locus (GenBank Accession No. AJ001300), the Pox3 gene locus (Pox3:GenBank Accession No. XP_(—)503244; or, Aco3: GenBank Accession No.AJ001301), the Δ12 desaturase gene locus (SEQ ID NO:28), the Lip1 genelocus (GenBank Accession No. Z50020) and/or the Lip2 gene locus (GenBankAccession No. AJ012632).

Advantageously, the Ura3 gene can be used repeatedly in combination with5-FOA selection (supra). More specifically, one can first knockout thenative Ura3 gene to produce a strain having a Ura-phenotype, whereinselection occurs based on 5-FOA resistance. Then, a cluster of multiplechimeric genes and a new Ura3 gene could be integrated into a differentlocus of the Yarrowia genome to thereby produce a new strain having aUra+ phenotype. Subsequent integration would produce a new Ura3- strain(again identified using 5-FOA selection), when the introduced Ura3 geneis knocked out. Thus, the Ura3 gene (in combination with 5-FOAselection) can be used as a selection marker in multiple rounds oftransformation and thereby readily permit genetic modifications to beintegrated into the Yarrowia genome in a facile manner.

For some applications, it will be useful to direct the instant proteinsto different cellular compartments (e.g., the acyl-CoA pool versus thephosphatidylcholine pool). For the purposes described herein, DHA may befound as free fatty acids or in esterified forms such as acylglycerols,phospholipids, sulfolipids or glycolipids. It is envisioned that thechimeric genes described above encoding polypeptides that permit DHAbiosynthesis may be further engineered to include appropriateintracellular targeting sequences.

Juretzek et al. (Yeast, 18:97-113 (2001)) note that the stability ofintegrated plasmid copy number in Yarrowia lipolytica is dependent onthe individual transformants, the recipient strain and the targetingplatform used. Thus, the skilled artisan will recognize that multipletransformants must be screened in order to obtain a strain displayingthe desired expression level and pattern. Such screening may beaccomplished by Southern analysis of DNA blots (Southern, J. Mol. Biol.98:503 (1975)), Northern analysis of mRNA expression (Kroczek, J.Chromatogr. Biomed. Appl., 618 (1-2):133-145 (1993)), Western analysisof protein expression, phenotypic analysis or GC analysis of the PUFAproducts.

In summary, each of the means described above is useful to increase theexpression of a particular gene product (e.g., a desaturase, elongase,acyltransferase) in Yarrowia lipolytica; and, one skilled in the art ofbiotechnology will readily be capable of selecting the most appropriatecombinations of methods to enable high production of DHA.

Pathway Engineering for Increased DHA Production

Although the methodology described above is useful to up-regulate theexpression of individual heterologous genes, the challenge of increasingDHA production in Yarrowia lipolytica is much more complex and mayrequire coordinated manipulation of various metabolic pathways.Manipulations in the PUFA biosynthetic pathway will be addressed first,followed by desirable manipulations in the TAG biosynthetic pathway andthe TAG degradation pathway.

As previously described, the construction of a Yarrowia lipolyticastrain producing greater than 5% DHA in the total oil fraction, or morepreferably greater than 10% DHA in the total oil fraction, or even morepreferably greater than 15-20% DHA in the total oil fraction, or mostpreferably greater than 25-30% DHA in the total oil fraction requires atleast the following genes: (1) for expression of the Δ6 desaturase/Δ6elongase pathway—a Δ6 desaturase, a C_(18/20)elongase, a Δ5 desaturase,either a Δ17 desaturase or a Δ15 desaturase (or both), a C_(20/22)elongase and a Δ4 desaturase; or, (2) at least the following genes forexpression of the Δ9 elongase/Δ8 desaturase pathway—a Δ9 elongase, a Δ8desaturase, a Δ5 desaturase, either a Δ17 desaturase or a Δ15 desaturase(or both), a C_(20/22) elongase and a Δ4 desaturase. In eitherembodiment, however, it may be desirable to additionally include a Δ9desaturase, a Δ12 desaturase, a C_(14/16) elongase and/or aC_(16/18)elongase in the host strain.

In some cases, it may prove advantageous to replace the native Yarrowialipolytica Δ12 desaturase with the Fusarium moniliforme Δ12 desaturase,since the latter shows increased percent substrate conversion (WO2005/047485). More specifically, although both Δ12 desaturases catalyzethe conversion of oleic acid to LA, the two enzymes differ in theiroverall specificity (which thereby affects each enzyme's percentsubstrate conversion). The Applicants have determined that the F.moniliforme Δ12 desaturase has a higher loading capacity of LA onto thesn-2 position of a phosphotidylcholine substrate (thereby facilitatingthe subsequent reaction by Δ6 desaturase) than the Y. lipolytica Δ12desaturase. On this basis, overexpression of the F. moniliforme Δ12desaturase in conjunction with a knockout of the Y. lipolytica Δ12desaturase may result in increased product for subsequent conversion toDHA.

In some embodiments, it may be useful to regulate the activity of a hostorganism's native DAG ATs to thereby enable manipulation of the percentof PUFAs within the lipids and oils of the Y. lipolytica host.

Specifically, since oil biosynthesis is expected to.compete withpolyunsaturation during oleaginy, it is possible to reduce or inactivatethe activity of an organism's one or more acyltransferases (e.g., PDATand/or DGAT1 and/or DGAT2), to thereby reduce the overall rate of oilbiosynthesis while concomitantly increasing the percent of PUFAs(relative to the total fatty acids) that are incorporated into the lipidand oil fractions. This results since polyunsaturation is permitted tooccur more efficiently; or, in other words, by down-regulating theactivity of specific DAG ATs, the substrate competition between oilbiosynthesis and polyunsaturation is reduced in favor ofpolyunsaturation during oleaginy.

One skilled in the art will have the skills necessary to elucidate theoptimum level of down-regulation and the means required to achieve suchinhibition. For example, in some preferred embodiments, it may bedesirable to manipulate the activity of a single DAG AT (e.g., create aDGAT1 knockout, while the activity of PDAT and DGAT2 are not altered).In alternate embodiments, the oleaginous organism comprises at total of“n” native DAG ATs and the activity of a total of “n-1” acyltransferasesare modified to result in a reduced rate of oil biosynthesis, while theremaining acyltransferase retains its wildtype activity. And, in somesituations, it may be desirable to manipulate the activity of all of thenative DAG ATs in some preferred oleaginous organisms, to achieve theoptimum rate of oil biosynthesis with respect to the rate ofpolyunsaturation.

In a similar manner, the Applicants hypothesize that expression ofheterologous acyltransferases in conjunction with knockouts of thecorresponding native Yarrowia lipolytica acyltransferase cansignificantly increase the overall DHA that is produced in the hostcells. Specifically, as suggested previously, heterologous GPAT, LPAAT,DGAT1, DGAT2, PDAT and LPCAT acyltransferases that have specificity forthose fatty acids that are C20 and greater could be preferred over thenative enzymes, since naturally produced PUFAs in Y. lipolytica arelimited to 18:2 fatty acids and the native enzymes may not efficientlycatalyze reactions with longer-chain fatty acids. Based on thisconclusion, the Applicants identified the genes encoding GPAT, LPAAT,DGAT1 and DGAT2 in M. alpina and expressed these genes in engineeredYarrowia hosts producing EPA, resulting in increased PUFA biosynthesis(Examples 17-20 herein). Subsequently, the activity of several of thenative acyltransferases (e.g., DGAT1 and DGAT2) in Y. lipolytica werediminished or knocked-out, as a means to reduce substrate competitionbetween the native and heterologous acyltransferase. Similar resultswould be expected in an engineered Yarrowia host producing DHA.

One must also consider manipulation of pathways and global regulatorsthat affect DHA production. For example, it is useful to increase theflow of carbon into the PUFA biosynthetic pathway by increasing theavailability of the precursors of longer chain saturated and unsaturatedfatty acids, such as palmitate (16:0) and stearic acid (18:0). Thesynthesis of the former is dependent on the activity of a C_(14/16)elongase, while the synthesis of the latter is dependent on the activityof a C_(16/18) elongase. Thus, over-expression of the native Yarrowialipolytica C_(14/16) elongase (SEQ ID NOs:97 and 98) substantiallyincreased the production of 16:0 and 16:1 fatty acids (22% increaserelative to control strains); similarly, over-expression of the nativeY. lipolytica C_(16/18) elongase (SEQ ID NOs:94 and 95) substantiallyincreased the production of 18:0, 18:1, 18:2 and 18:3 fatty acids (18%increase relative to control strains) and reduced the accumulation ofC₁₆ fatty acids (22% decrease relative to control strains). Of course,as demonstrated herein and as suggested by the work of Inagaki, K. etal. (Biosci. Biotech. Biochem. 66(3):613-621 (2002)), in someembodiments of the present invention it may be useful to ω-express aheterologous C_(16/18) elongase (e.g., from Rattus norvegicus [GenBankAccession No. AB071986; SEQ ID NOs:83 and 84 herein] and/or from M.alpina [SEQ ID NO:86 and 87]. Thus, although a Y. lipolytica host strainmust minimally be manipulated to express either: (1) a Δ6 desaturase, aC_(18/20) elongase, a Δ5 desaturase, either a Δ17 desaturase or a Δ15desaturase (or both), a C_(20/22) elongase and a Δ4 desaturase; or, (2)a Δ9 elongase, a Δ8 desaturase, a Δ5 desaturase, either a Δ17 desaturaseor a Δ15 desaturase (or both), a C_(20/22) elongase and a Δ4 desaturasefor DHA biosynthesis, in further preferred embodiments the host strainadditionally includes at least one of the following: a Δ9 desaturase, aΔ12 desaturase, a C_(14/16)elongase and/or a C_(16/18) elongase.

In another preferred embodiment, those pathways that affect fatty aciddegradation and TAG degradation can be modified in the Yarrowialipolytica of the present invention, to mimimize the degradation of DHAthat accumulates in the cells in either the acyl-CoA pool or in the TAGfraction. These pathways are represented by the acyl-CoA oxidase andlipase genes, respectively. More specifically, the acyl-CoA oxidases (EC1.3.3.6) catalyze a peroxisomal β-oxidation reaction wherein each cycleof degradation yields an acetyl-CoA molecule and a fatty acid that istwo carbon atoms shorter than the fatty acid substrate. Five acyl-CoAoxidase isozymes are present in Yarrowia lipolytica, encoded by thePOX1, POX2, POX3, POX4 and POX5 genes (also known as the Aco1, Aco2,Aco3, Aco4 and Aco5 genes), corresponding to GenBank Accession Nos.AJ001299-AJ001303, respectively (see also corresponding GenBankAccession Nos. XP_(—)504703, XP_(—)505264, XP_(—)503244, XP_(—)504475and XP_(—)502199). Each of the isozymes has a different substratespecificity; for example, the POX3 gene encodes an acyl-CoA oxidase thatis active against short-chain fatty acids, whereas the POX2 gene encodesan acyl-CoA oxidase that is active against longer-chain fatty acids(Wang H. J., et al. J. Bacteriol., 181:5140-5148 (1999)). It iscontemplated that the activity of any one of these genes could bereduced or eliminated, to thereby modify peroxisomal β-oxidation in thehost cell of the invention in a manner that could be advantageous to thepurposes herein. Finally, to avoid any confusion, the Applicants willrefer to the acyl-CoA oxidases as described above as POX genes, althoughthis terminology can be used interchangeably with the Aco genenomeclature, according to some publicly available literature.

Similarly, several lipases (EC 3.1.1.3) have been detected in Y.lipolytica, including intracellular, membrane-bound and extracellularenzymes (Choupina, A., et al. Curr. Genet. 35:297 (1999); Pignede, G.,et al. J. Bacteriol. 182:2802-2810 (2000)). For example, Lip1 (GenBankAccession No. Z50020) and Lip3 (GenBank Accession No. AJ249751) areintracellular or membrane bound, while Lip2 (GenBank Accession No.AJ012632) encodes an extracellular lipase. Each of these lipases aretargets for disruption, since the enzymes catalyze the reaction whereinTAG and water are degraded directly to DAG and a fatty acid anion.

In a further alternate embodiment, the activity of severalphospholipases can be manipulated in the preferred host strain ofYarrowia lipolytica. Phospholipases play a critical role in thebiosynthesis and degradation of membrane lipids. More specifically, theterm “phospholipase” refers to a heterogeneous group of enzymes thatshare the ability to hydrolyze one or more ester linkage inglycerophospholipids. Although all phospholipases target phospholipidsas substrates, each enzyme has the ability to cleave a specific esterbond. Thus, phospholipase nomeclature differentitates individualphospholipases and indicates the specific bond targeted in thephospholipid molecule. For example, phospholipase A₁ (PLA₁) hydrolyzesthe fatty acyl ester bond at the sn-1 position of the glycerol moiety,while phospholipase A₂ (PLA₂) removes the fatty acid at the sn-2position of this molecule. The action of PLA₁ (EC 3.1.1.32) and PLA₂ (EC3.1.1.4) results in the accumulation of free fatty acids and 2-acyllysophospholipid or 1-acyl lysophospholipid, respectively. PhospholipaseC (PLC) (EC 3.1.4.3) hydrolyzes the phosphodiester bond in thephospholipid backbone to yield 1,2-DAG and, depending on the specificphospholipid species involved, phosphatidylcholine,phosphatidylethanolamine, etc. (e.g., PLC₁ is responsible for thereaction: 1-phosphatidyl-1 D-myo-inositol4,5-bisphosphate+H₂O=1D-myo-inositol 1,4,5-trisphosphate+DAG; ISC1encodes an inositol phosphosphingolipid-specific phospholipase C [Sawai,H., et al. J. Biol. Chem. 275, 39793-39798 (2000)]). The secondphosphodiester bond is cleaved by phospholipase D (PLD) (EC 3.1.4.4) toyield phosphatidic acid and choline or ethanolamine, again depending onthe phospholipid class involved. Phospholipase B (PLB) has thecapability of removing both sn-1 and sn-2 fatty acids and is unique inhaving both hydrolase (wherein the enzyme cleaves fatty acids from bothphospholipids [PLB activity] and lysophospholipids [lysophospholipaseactivity] for fatty acid release) and lysophospholipase-transacylaseactivities (wherein the enzyme can produce phospholipid by transferringa free fatty acid to a lysophospholipid). It may be useful tooverexpress one or more of these phopsholipases, in order to increasethe concentration of DHA that accumulates in the total oil fraction ofthe transformant Yarrowia host cells. It is hypothesized that thisresult will be observed because the phospholipases release acyl groupsfrom PC into the CoA pool either for elongation or incorporation intotriglycerides.

In another alternate embodiment, those enzymes in the CDP-cholinepathway responsible for phosphatidylcholine (PC) biosynthesis can alsobe manipulated in the preferred host strain of Yarrowia lipolytica, as ameans to increase overall DHA biosynthesis. The utility of thistechnique has been demonstrated by the overexpression of the Y.lipolytica CPT1 gene encoding diacylglycerol cholinephosphotransferase(EC 2.7.8.2), thereby resulting in increased EPA biosynthesis in anengineered strain of Y. lipolytica. One skilled in the art will befamiliar with the PC biosynthetic pathway and recognize otherappropriate candidate enzymes.

Although methods for manipulating biochemical pathways such as thosedescribed above are well known to those skilled in the art, an overviewof some techniques for reducing or eliminating the activity of a nativegene will be briefly presented below. These techniques would be usefulto down-regulate the activity of the native Yarrowia lipolytica Δ12desaturase, GPAT, LPAAT, DGAT1, DGAT2, PDAT, LPCAT, acyl-CoA oxidase 2(Aco2 or Pox2), acyl-CoA oxidase 3 (Aco3 or Pox3) and/or lipase genes,as discussed above.

Although one skilled in the art will be well equipped to ascertain themost appropriate technique to be utilized to reduce or eliminate theactivity of a native gene, in general, the endogenous activity of aparticular gene can be reduced or eliminated by, for example: 1.)disrupting the gene through insertion, substitution and/or deletion ofall or part of the target gene; 2.) providing a cassette fortranscription of antisense sequences to the gene's transcriptionproduct; 3.) using a host cell which naturally has [or has been mutatedto have] little or none of the specific gene's activity; 4.)over-expressing a mutagenized hereosubunit (i.e., in an enzyme thatcomprises two or more hereosubunits), to thereby reduce the enzyme'sactivity as a result of the “dominant negative effect”; and 5.) usingiRNA technology. In some cases, inhibition of undesired gene pathwayscan also be accomplished through the use of specific inhibitors (e.g.,desaturase inhibitors such as those described in U.S. Pat. No.4,778,630).

For gene disruption, a foreign DNA fragment (typically a selectablemarker gene, but optionally a chimeric gene or chimeric gene clusterconveying a desirable phenotype upon expression) is inserted into thestructural gene to be disrupted in order to interrupt its codingsequence and thereby functionally inactivate the gene. Transformation ofthe disruption cassette into the host cell results in replacement of thefunctional native gene by homologous recombination with thenon-functional disrupted gene (see, for example: Hamilton et al. J.Bacteriol. 171:4617-4622 (1989); Balbas et al. Gene 136:211-213 (1993);Gueldener et al. Nucleic Acids Res. 24:2519-2524 (1996); and Smith etal. Methods Mol. Cell. Biol. 5:270-277(1996)).

Antisense technology is another method of down-regulating genes when thesequence of the target gene is known. To accomplish this, a nucleic acidsegment from the desired gene is cloned and operably linked to apromoter such that the anti-sense strand of RNA will be transcribed.This construct is then introduced into the host cell and the antisensestrand of RNA is produced. Antisense RNA inhibits gene expression bypreventing the accumulation of mRNA that encodes the protein ofinterest. The person skilled in the art will know that specialconsiderations are associated with the use of antisense technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of antisense genes may require the use of differentchimeric genes utilizing different regulatory elements known to theskilled artisan.

Although targeted gene disruption and antisense technology offereffective means of down-regulating genes where the sequence is known,other less specific methodologies have been developed that are notsequence-based (e.g., mutagenesis via UV radiation/chemical agents oruse of transposable elements/transposons; see WO 04/101757).

In alternate embodiments, the endogenous activity of a particular genecan be reduced by manipulating the regulatory sequences controlling theexpression of the protein. As is well known in the art, the regulatorysequences associated with a coding sequence include transcriptional andtranslational “control” nucleotide sequences located upstream (5′non-coding sequences), within, or downstream (3′ non-coding sequences)of the coding sequence, and which influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence. Thus, manipulation of a particular gene's regulatory sequencesmay refer to manipulation of the gene's promoters, translation leadersequences, introns, enhancers, initiation control regions,polyadenylation recognition sequences, RNA processing sites, effectorbinding sites and stem-loop structures. Thus, for example, the promoterof a DAG AT could be deleted or disrupted, in order to down-regulate theDAG AT's expression and thereby achieve a reduced rate of lipid and oilbiosynthesis. Alternatively, the native promoter driving expression of aDAG AT could be substituted with a heterologous promoter havingdiminished promoter activity with respect to the native promoter.Methods useful for manipulating regulatory sequences are well known tothose skilled in the art.

In summary, using the teachings provided herein, transformant oleaginousmicrobial hosts will produce at least about 5% DHA in the total lipids,more preferably at least about 10% DHA in the total lipids, morepreferably at least about 15% DHA in the total lipids, more preferablyat least about 20% DHA in the total lipids and most preferably at leastabout 25-30% DHA in the total lipids.

Fermentation Processes for DHA Production

The transformed microbial host cell is grown under conditions thatoptimize expression of chimeric genes (e.g., encoding desaturases,elongases, acyltransferases, etc.) and produce the greatest and the mosteconomical yield of DHA. In general, media conditions that may beoptimized include the type and amount of carbon source, the type andamount of nitrogen source, the carbon-to-nitrogen ratio, the oxygenlevel, growth temperature, pH, length of the biomass production phase,length of the oil accumulation phase and the time of cell harvest.Yarrowia lipolytica are generally grown in complex media (e.g., yeastextract-peptone-dextrose broth (YPD)) or a defined minimal media thatlacks a component necessary for growth and thereby forces selection ofthe desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCOLaboratories, Detroit, Mich.)).

Fermentation media in the present invention must contain a suitablecarbon source. Suitable carbon sources may include, but are not limitedto: monosaccharides (e.g., glucose, fructose), disaccharides (e.g.,lactose, sucrose), oligosaccharides, polysaccharides (e.g., starch,cellulose or mixtures thereof, sugar alcohols (e.g., glycerol) ormixtures from renewable feedstocks (e.g., cheese whey permeate,cornsteep liquor, sugar beet molasses, barley malt). Additionally,carbon sources may include alkanes, fatty acids, esters of fatty acids,monoglycerides, diglycerides, triglycerides, phospholipids and variouscommercial sources of fatty acids including vegetable oils (e.g.,soybean oil) and animal fats. Additionally, the carbon source mayinclude one-carbon sources (e.g., carbon dioxide, methanol,formaldehyde, formate and carbon-containing amines) for which metabolicconversion into key biochemical intermediates has been demonstrated.Hence it is contemplated that the source of carbon utilized in thepresent invention may encompass a wide variety of carbon-containingsources. Although all of the above mentioned carbon sources and mixturesthereof are expected to be suitable in the present invention, preferredcarbon sources are sugars and/or fatty acids. Most preferred is glucoseand/or fatty acids containing between 10-22 carbons.

Nitrogen may be supplied from an inorganic (e.g., (NH₄)₂SO₄) or organic(e.g., urea or glutamate) source. In addition to appropriate carbon andnitrogen sources, the fermentation media must also contain suitableminerals, salts, cofactors, buffers, vitamins and other components knownto those skilled in the art suitable for the growth of the oleaginousyeast and promotion of the enzymatic pathways necessary for DHAproduction. Particular attention is given to several metal ions (e.g.,Mn⁺², Co⁺², Zn⁺², Mg⁺²) that promote synthesis of lipids and PUFAs(Nakahara, T. et al., Ind. Appl. Single Cell Oils, D. J. Kyle and R.Colin, eds. pp 61-97 (1992)).

Preferred growth media in the present invention are common commerciallyprepared media, such as Yeast Nitrogen Base (DIFCO Laboratories,Detroit, Mich.). Other defined or synthetic growth media may also beused and the appropriate medium for growth of Yarrowia lipolytica willbe known by one skilled in the art of microbiology or fermentationscience. A suitable pH range for the fermentation is typically betweenabout pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.0 is preferred as therange for the initial growth conditions. The fermentation may beconducted under aerobic or anaerobic conditions, wherein microaerobicconditions are preferred.

Typically, accumulation of high levels of PUFAs in oleaginous yeastcells requires a two-stage process, since the metabolic state must be“balanced” between growth and synthesis/storage of fats. Thus, mostpreferably, a two-stage fermentation process is necessary for theproduction of DHA in Yarrowia lipolytica. This approach is described inWO 2004/101757, as are various suitable fermentation process designs(i.e., batch, fed-batch and continuous) and considerations duringgrowth.

Purification of and Processing of DHA

PUFAs, including DHA, may be found in the host microorganism as freefatty acids or in esterified forms such as acylglycerols, phospholipids,sulfolipids or glycolipids, and may be extracted from the host cellthrough a variety of means well-known in the art. One review ofextraction techniques, quality analysis and acceptability standards foryeast lipids is that of Z. Jacobs (Critical Reviews in Biotechnology,12(5/6):463-491 (1992)). A brief review of downstream processing is alsoavailable by A. Singh and O. Ward (Adv. Appl. Microbiol., 45:271-312(1997)).

In general, means for the purification of DHA and other PUFAs mayinclude extraction with organic solvents, sonication, supercriticalfluid extraction (e.g., using carbon dioxide), saponification andphysical means such as presses, or combinations thereof. One is referredto the teachings of WO 2004/101757 for additional details.

Oils containing DHA that have been refined and/or purified can behydrogenated, to thereby result in fats with various melting propertiesand textures. Many processed fats, including spreads, confectionaryfats, hard butters, margarines, baking shortenings, etc., requirevarying degrees of solidity at room temperature and can only be producedthrough alteration of the source oil's physical properties. This is mostcommonly achieved through catalytic hydrogenation.

Hydrogenation is a chemical reaction in which hydrogen is added to theunsaturated fatty acid double bonds with the aid of a catalyst such asnickel. Hydrogenation has two primary effects. First, the oxidativestability of the oil is increased as a result of the reduction of theunsaturated fatty acid content.′ Second, the physical properties of theoil are changed because the fatty acid modifications increase themelting point resulting in a semi-liquid or solid fat at roomtemperature.

There are many variables which affect the hydrogenation reaction andwhich, in turn, alter the composition of the final product. Operatingconditions including pressure, temperature, catalyst type andconcentration, agitation and reactor design are among the more importantparameters which can be controlled. Selective hydrogenation conditionscan be used to hydrogenate the more unsaturated fatty acids inpreference to the less unsaturated ones. Very light or brushhydrogenation is often employed to increase stability of liquid oils.Further hydrogenation converts a liquid oil to a physically solid fat.The degree of hydrogenation depends on the desired performance andmelting characteristics designed for the particular end product. Liquidshortenings, used in the manufacture of baking products, solid fats andshortenings used for commercial frying and roasting operations, and basestocks for margarine manufacture are among the myriad of possible oiland fat products achieved through hydrogenation. A more detaileddescription of hydrogenation and hydrogenated products can be found inPatterson, H. B. W., Hydrogenation of Fats and Oils: Theory andPractice. The American Oil Chemists' Society, 1994.

DHA-Producing Strains of Y. lipolytica for Use in Foodstuffs

The market place currently supports a large variety of food and feedproducts, incorporating ω-3 and/or ω-6 fatty acids (particularly ARA,EPA and DHA). It is contemplated that the yeast microbial oils of theinvention comprising DHA will function in food and feed products toimpart the health benefits of current formulations.

Microbial oils containing ω-3 and/or ω-6 fatty acids produced by theyeast hosts described herein will be suitable for use in a variety offood and feed products including, but not limited to food analogs, meatproducts, cereal products, baked foods, snack foods, and a dairyproducts. Additionally the present microbial oils may be used informulations to impart health benefit in medical foods including medicalnutritionals, dietary supplements, infant formula as well aspharmaceutical products. One of skill in the art of food processing andfood formulation will understand how the amount and composition of themicrobial oil may be added to the food or feed product. Such an amountwill be referred to herein as an “effective” amount and will depend onthe food or feed product, the diet that the product is intended tosupplement or the medical condition that the medical food or medicalnutritional is intended to correct or treat.

Food analogs can be made using processes well known to those skilled inthe art. There can be mentioned meat analogs, cheese analogs, milkanalogs and the like. Meat analogs made from soybeans contain soyprotein or tofu and other ingredients mixed together to simulate variouskinds of meats. These meat alternatives are sold as frozen, canned ordried foods. Usually, they can be used the same way as the foods theyreplace. Examples of meat analogs include, but are not limited to: hamanalogs, sausage analogs, bacon analogs, and the like.

Food analogs can be classified as imitation or substitutes depending ontheir functional and compositional characteristics. For example, animitation cheese need only resemble the cheese it is designed toreplace. However, a product can generally be called a substitute cheeseonly if it is nutritionally equivalent to the cheese it is replacing andmeets the minimum compositional requirements for that cheese. Thus,substitute cheese will often have higher protein levels than imitationcheeses and be fortified with vitamins and minerals.

Milk analogs or nondairy food products include, but are not limited to:imitation milks and nondairy frozen desserts (e.g., those made fromsoybeans and/or soy protein products).

Meat products encompass a broad variety of products. In the UnitedStates “meat” includes “red meats” produced from cattle, hogs and sheep.In addition to the red meats there are poultry items which includechickens, turkeys, geese, guineas, ducks and the fish and shellfish.There is a wide assortment of seasoned and processed meat products:fresh, cured and fried, and cured and cooked. Sausages and hot dogs areexamples of processed meat products. Thus, the term “meat products” asused herein includes, but is not limited to, processed meat products.

A cereal food product is a food product derived from the processing of acereal grain. A cereal grain includes any plant from the grass familythat yields an edible grain (seed). The most popular grains are barley,corn, millet, oats, quinoa, rice, rye, sorghum, triticale, wheat andwild rice.

Examples of a cereal food product include, but are not limited to: wholegrain, crushed grain, grits, flour, bran, germ, breakfast cereals,extruded foods, pastas, and the like.

A baked goods product comprises any of the cereal food productsmentioned above and has been baked or processed in a manner comparableto baking, i.e., to dry or harden by subjecting to heat. Examples of abaked good product include, but are not limited to: bread, cakes,doughnuts, bars, pastas, bread crumbs, baked snacks, mini-biscuits,mini-crackers, mini-cookies, and mini-pretzels. As was mentioned above,oils of the invention can be used as an ingredient.

A snack food product comprises any of the above or below described foodproducts.

A fried food product comprises any of the above or below described foodproducts that has been fried.

The beverage can be in a liquid or in a dry powdered form.

For example, there can be mentioned non-carbonated drinks; fruit juices,fresh, frozen, canned or concentrate; flavored or plain milk drinks,etc. Adult and infant nutritional formulas are well known in the art andcommercially available (e.g., Similac®, Ensure®, Jevity®, and Alimentum®from Ross Products Division, Abbott Laboratories). Infant formulas areliquids or reconstituted powders fed to infants and young children. Theyserve as substitutes for human milk. Infant formulas have a special roleto play in the diets of infants because they are often the only sourceof nutrients for infants. Although breast-feeding is still the bestnourishment for infants, infant formula is a close enough second thatbabies not only survive but thrive. Infant formula is becoming more andmore increasingly close to breast milk.

A dairy product is a product derived from milk. A milk analog ornondairy product is derived from a source other than milk, for example,soymilk as was discussed above. These products include, but are notlimited to: whole milk, skim milk, fermented milk products such asyogurt or sour milk, cream, butter, condensed milk, dehydrated milk,coffee whitener, coffee creamer, ice cream, cheese, etc.

Additional food products into which the DHA-containing oils of theinvention could be included are, for example: chewing gums, confectionsand frostings, gelatins and puddings, hard and soft candies, jams andjellies, white granulated sugar, sugar substitutes, sweet sauces,toppings and syrups, and dry-blended powder mixes.

Health Food Products, and Pharmaceuticals

A health food product is any food product that imparts a health benefitand include functional foods, medical foods, medical nutritionals anddietary supplements. Additionally, microbial oils of the invention maybe used in standard pharmaceutical compositions. The present engineeredstrains of Yarrowia lipolytica or the microbial oils produced therefromcomprising DHA could readily be incorporated into the any of the abovementioned food products, to thereby produce e.g., a functional ormedical food. For example more concentrated formulations comprising DHAinclude capsules, powders, tablets, softgels, gelcaps, liquidconcentrates and emulsions which can be used as a dietary supplement inhumans or animals other than humans.

Use in Dietary Supplements

More concentrated formulations comprising DHA include capsules, powders,tablets, softgels, gelcaps, liquid concentrates and emulsions which canbe used as a dietary supplement in humans or animals other than humans.In particular, the DHA-oil of the present invention is particularlysuitable for incorporation into dietary supplements such as infantformulas or baby food.

Infant formulas are liquids or reconstituted powders fed to infants andyoung children. “Infant formula” is defined herein as an externalnutritional product which can be substituted for human breast milk infeeding infants and typically is composed of a desired percentage of fatmixed with desired percentages of carbohydrates and proteins in anaqueous solution (e.g., see U.S. Pat. No. 4,670,285). Based on theworldwide composition studies, as well as levels specified by expertgroups, average human breast milk typically contains about 0.20% to0.40% of total fatty acids (assuming about 50% of calories from fat);and, generally the ratio of DHA to ARA would range from about 1:1 to 1:2(see, e.g., formulations of Enfamil LIPIL™ [Mead Johnson & Company] andSimilac Advance™ [Ross Products Division, Abbott Laboratories]). Infantformulas have a special role to play in the diets of infants becausethey are often the only source of nutrients for infants; and, althoughbreast-feeding is still the best nourishment for infants, infant formulais a close enough second that babies not only survive but thrive.

Use in Animal Feeds

Animal feeds are generically defined herein as products intended for useas feed or for mixing in feed for animals other than humans. And, as wasmentioned above, the DHA-comprising oils of the invention can be used asan ingredient in various animal feeds.

More specifically, although not limited therein, it is expected that theoils of the invention can be used within pet food products, ruminant andpoultry food products and aquacultural food products. Pet food productsare those products intended to be fed to a pet [e.g., a dog, cat, bird,reptile, rodent]; these products can include the cereal and health foodproducts above, as well as meat and meat byproducts, soy proteinproducts, grass and hay products (e.g., alfalfa, timothy, oat or bromegrass, vegetables). Ruminant and poultry food products are those whereinthe product is intended to be fed to e.g., turkeys, chickens, cattle andswine. As with the pet foods above, these products can include cerealand health food products, soy protein products, meat and meatbyproducts, and grass and hay products as listed above. And,aquacultural food products (or “aquafeeds”) are those products intendedto be used in aquafarming which concerns the propagation, cultivation orfarming of aquatic organisms and/or animals in fresh or marine waters.

It is contemplated that the present engineered strains of Yarrowialipolytica that are producing high concentrations of ARA, EPA and/or DHAwill be especially useful to include in most animal feed formulations.In addition to providing necessary ω-3 and/or ω-6 PUFAs, the yeastitself is a useful source of protein and other feed nutrients (e.g.,vitamins, minerals, nucleic acids, complex carbohydrates, etc.) that cancontribute to overall animal health and nutrition, as well as increase aformulation's palatablility. More specifically, Yarrowia lipolytica(ATCC #20362) has the following approximate chemical composition, as apercent relative to the dry cell weight: 35% protein, 40% lipid, 10%carbohydrate, 5% nucleic acids, 5% ash and 5% moisture. Furthermore,within the carbohydrate fraction, β-glucans comprise approximately 45.6mg/g, mannans comprise approximately 11.4 mg/g, and chitin comprisesapproximately 52.6 mg/g (while trehalose is a minor component[approximately 0.7 mg/g]).

A considerable body of literature has examined the immuno-modulatingeffects of β-glucans, mannans and chit in. The means by which β-glucans,the primary constituents of bacterial and fungal cell walls, stimulatenon-specific immunity (i.e., “immunostimulant effects”) to therebyimprove health of aqua culture species, pets and farm animals and humansare best studied, although both chit in and mannans are similarlyrecognized as useful immunostimulants. Simplistically, an overallenhancement of immune response can be achieved by the use of β-glucans,since these β-1,3-D-polyglucose molecules stimulate the production ofwhite blood cells (e.g., macrophages, neutrophils and monocytes) in anon-specific manner to thereby enable increased sensitivity and defenseagainst a variety of pathogenic antigens or environmental stressors.More specifically, numerous studies have demonstrated that β-glucans:convey enhanced protection against viral, bacterial, fungal andparasitic infections; exert an adjuvant effect when used in conjunctionwith antibiotics and vaccines; enhance wound healing; counter damageresulting from free radicals; enhance tumor regression; modulatetoxicity of bacterial endotoxins; and strengthen mucosal immunity(reviewed in Raa, J. et al., Norwegian Beta Glucan Research, ClinicalApplications of Natural Medicine. Immune: Depressions Dysfunction &Deficiency (1990)). A sample of current literature documenting theutility of yeast β-glucans, mannans and chitins in both traditionalanimal husbandry and within the aquacultural sector include: L. A. Whiteet al. (J. Anim. Sci. 80:2619-2628 (2002)), supplementation in weanlingpigs; K. S. Swanson et al. (J. Nutr. 132:980-989 (2002)),supplementation in dogs; J. Ortuño et al. (Vet. Immunol. Immonopath.85:41-50 (2002)), whole Saccharomyces cerevisiae administered togilthead seabream; A. Rodriguez et al. (Fish Shell. Immuno. 16:241-249(2004)), whole Mucor circinelloides administered to gilthead seabream;M. Bagni et al. (Fish Shell. Immuno. 18:311-325 (2005)), supplementationof sea bass with a yeast extract containing β-glucans; J. Raa (In:Cruz-Suárez, L. E., Ricque-Marie, D., Tapia-Salazar, M., Olvera-Novoa,M. A. y Civera-Cerecedo, R., (Eds.). Avances en Nutrición Acuicola V.Memorias del V Simposium Internacional de Nutrición Acuicola. 19-22Noviembre, 2000. Mérida, Yucatán, Mexico), a review of the use ofimmune-stimulants in fish and shellfish feeds.

Based on the unique protein:lipid:carbohydrate composition of Yarrowialipolytica, as well as unique complex carbohydrate profile (comprisingan approximate 1:4:4.6 ratio of mannan:β-glucans:chit in), it iscontemplated that the genetically engineered yeast cells of the presentinvention (or portions thereof) would be a useful additive to animalfeed formulations (e.g., as whole [lyophilized] yeast cells, as purifiedcells walls, as purified yeast carbohydrates or within various otherfractionated forms).

With respect to the aqua culture industry, an increased understanding ofthe nutritional requirements for various fish species and technologicaladvances in feed manufacturing have allowed the development and use ofmanufactured or artificial diets (formulated feeds) to supplement or toreplace natural feeds in the aqua culture industry. In general, however,the general proportions of various nutrients included in aqua culturefeeds for fish include (with respect to the percent by dry diet): 32-45%proteins, 4-28% fat (of which at least 1-2% are ω-3 and/or ω-6 PUFAs),10-30% carbohydrates, 1.0-2.5% minerals and 1.0-2.5% vitamins. A varietyof other ingredients may optionally be added to the formulation. Theseinclude: (1) carotenoids, particularly for salmonid and ornamental“aquarium” fishes, to enhance flesh and skin coloration, respectively;(2) binding agents, to provide stability to the pellet and reduceleaching of nutrients into the water (e.g., beef heart, starch,cellulose, pectin, gelatin, gum arabic, locust bean, agar, carageeninand other alginates); (3) preservatives, such as antimicrobials andantioxidants, to extend the shelf-life of fish diets and reduce therancidity of the fats (e.g., vitamin E, butylated hydroxyanisole,butylated hydroxytoluene, ethoxyquin, and sodium and potassium salts ofpropionic, benzoic or sorbic acids); (4) chemoattractants andflavorings, to enhance feed palatability and its intake; and, (5) otherfeedstuffs. These other feedstuffs can include such materials as fiberand ash (for use as a filler and as a source of calcium and phosphorus,respectively) and vegetable matter and/or fish or squid meal (e.g.,live, frozen or dried algae, brine shrimp, rotifers or otherzooplankton) to enhance the nutritional value of the diet and increaseits acceptance by the fish. Nutrient Requirements of Fish (NationalResearch Council, National Academy: Washington D.C., 1993) providesdetailed descriptions of the essential nutrients for fish and thenutrient content of various ingredients.

The manufacture of aquafeed formulations requires consideration of avariety of factors, since a complete diet must be nutritionallybalanced, palatable, water stable, and have the proper size and texture.With regard to nutrient composition of aquafeeds, one is referred to:Handbook on Ingredients for Aquaculture Feeds (Hertrampf, J. W. and F.Piedad-Pascual. Kluwer Academic: Dordrecht, The Netherlands, 2000) andStandard Methods for the Nutrition and Feeding of Farmed Fish and Shrimp(Tacon, A. G. J. Argent Laboratories: Redmond, 1990). In general, feedsare formulated to be dry (i.e., final moisture content of 6-10%),semi-moist (i.e., 35-40% water content) or wet (i.e., 50-70% watercontent). Dry feeds include the following: simple loose mixtures of dryingredients (i.e., “mash” or “meals”); compressed pellets, crumbles orgranules; and flakes. Depending on the feeding requirements of the fish,pellets can be made to sink or float. Semi-moist and wet feeds are madefrom single or mixed ingredients (e.g., trash fish or cooked legumes)and can be shaped into cakes or balls.

It is contemplated that the present engineered strains of Yarrowialipolytical that are producing high concentrations of DHA will beespecially useful to include in most aqua culture feeds. In addition toproviding necessary ω-6 PUFAs, the yeast itself is a useful source ofprotein that can increase the formulation's palatablility. In alternateembodiments, the oils produced by the present strains of Y. lipolyticacould be introduced directly into the aqua culture feed formulations,following extraction and purification from the cell mass.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention demonstrates the synthesis of up to 5.6% DHA inthe total lipid fraction of the oleaginous yeast, Yarrowia lipolytica.As shown in FIG. 5, numerous strains of Y. lipolytica were created byintegrating various genes into.wildtype ATCC #20362 Y. lipolytica,wherein each transformant strain was capable of producing differentamounts of PUFAs (including DHA). The complete lipid profile of somerepresentative transformant organisms are shown below in Table 10.Elongases are identified using the abbreviated nomenclature as follows:C₁₈EL1 refers to a high affinity C_(18/20)elongase (ELO1); C₁₈EL2 refersto a C_(18/20)elongase (ELO2); C₁₆EL refers to a C_(16/18) elongase; andC₂₀EL refers to a C_(20/22) elongase. Fatty acids are identified as16:0, 16:1, 18:0, 18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETA,EPA, DPA and DHA; and the composition of each is presented as a % of thetotal fatty acids. “Lipid % dcw” represents the percentage of lipids inthe cell, as measured by dry cell weight.

TABLE 10 Lipid Profile Of Representative Yarrowia lipolytica StrainsExpressing The ω-6 Δ6 Desaturase/Δ6 Elongase Pathway Number Of GenesAdded C₁₈ C₁₈ C₁₆ C₂₀ Gene Fatty Acid Content Strain Δ12 Δ6 EL1 EL2 Δ5Δ17 EL Δ4 EL KOs 16:0 16:1 18:0 18:1 M4 1 1 1 1 — — — — — Ura 15 4 2 5EU 1 1 1 1 2 3 — — — Aco3 19 10.3 2.3 15.8 Ura Y2072 2 1 2 1 4 3 1 — —Aco3 7.6 4.1 2.2 16.8 Δ12 Y2089 3 2 3 1 5 3 2 — — Aco3 7.9 3.4 2.5 9.9Δ12 Y2098 3 2 3 1 5 3 2 — — Aco3 8.4 1.3 2 7.2 Δ12 Y3000 3 2 3 1 5 3 3 11 Aco3 5.9 1.2 5.5 7.7 Δ12 Fatty Acid Content Lipid % Strain 18:2 GLADGLA ARA ETA EPA DPA DHA dcw M4 27 35 8 0 0 0 — — — EU 12 18.7 5.7 0.2 310.3 — — 36 Y2072 13.9 27.8 3.7 1.7 2.2 15 — — — Y2089 14.3 37.5 2.5 1.81.6 17.6 — — — Y2098 11 24.6 4.3 1.2 2.4 25.6 — — — Y3000 11.7 30.1 2.61.2 1.2 4.7 18.3 5.6 —

As seen in the Table above, the strain producing DHA comprised thegenetic modifications described below (wherein complete details areprovided in the Examples):

-   -   (1) Expression of 2 copies of a Fusarium moniliforme Δ12        desaturase, within FBA::F.Δ12::LIP2 and TEF::F.Δ12::PEX16        chimeric genes;    -   (2) Expression of 1 copy of a Mortierella isabellina Δ12        desaturase, within a FBAIN::M.D12::PEX20 chimeric gene;    -   (3) Expression of 2 copies of a synthetic Δ6 desaturase gene        (codon-optimized for expression in Y. lipolytica) derived from a        Mortierella alpina Δ6 desaturase, within TEF::Δ6S::LIP1 and        FBAIN::Δ6S::LIP1 chimeric genes;    -   (4) Expression of 2 copies of a Mortierella alpina Δ5        desaturase, within FBAIN::MAΔ5S::PEX20 and TEF::MAΔ5S::LIP1        chimeric genes;    -   (5) Expression of 2 copies of a synthetic Δ5 desaturase gene        (codon-optimized for expression in Y. lipolytica) derived from a        Homo sapiens Δ5 desaturase, within TEF::H.D5S::PEX16 and        GPAT::H.D5S::PEX20 chimeric genes;    -   (6) Expression of 1 copy of a synthetic Δ5 desaturase gene        (codon-optimized for expression in Y. lipolytica) derived from a        Isochrysis galbana Δ5 desaturase, within a TEF::I.D5S::PEX20        chimeric gene;    -   (7) Expression of 3 copies of a synthetic Δ17 desaturase gene        (codon-optimized for expression in Y. lipolytica) derived from a        Saprolegnia diclina Δ17 desaturase, within FBAIN::Δ17S::LIP2,        TEF::Δ17S::PEX20 and FBAINm::Δ17S::PEX16 chimeric genes;    -   (8) Expression of 3 copies of a synthetic high affinity C18/20        elongase gene (codon-optimized for expression in Y. lipolytica)        derived from a Mortierella alpina high affinity elongase, within        FBAIN::EL1S::PEX20, GPAT::EL1S::XPR and GPDIN::EL1S::LIP2        chimeric genes;    -   (9) Expression of 1 copy of a synthetic C_(18/20) elongase gene        (codon-optimized for expression in Y. lipolytica) derived from a        Thraustochytrium aureum elongase, within a TEF::EL2S::XPR        chimeric gene;    -   (10) Expression of 3 copies of a synthetic C16/18 elongase gene        (codon-optimized for expression in Y. lipolytica) derived from a        Rattus norvegicus rELO gene, within TEF::rELO2S::PEX20 chimeric        genes;    -   (11) Expression of 1 copy of a synthetic Δ4 desaturase gene        (codon-optimized for expression in Y. lipolytica) derived from a        Thraustochytrium aureum Δ4 desaturase gene, within a        YAT1::Δ4S::Pex16 chimeric gene;    -   (12) Expression of 1 copy of a synthetic C_(20/22) elongase gene        (codon-optimized for expression in Y. lipolytica) derived from a        Ostreococcus tauri C_(20/22) elongase gene, within a        FBAIN::OtE2S::Oct chimeric gene;    -   (13) Disruption of a native Y. lipolytica Ura3 gene encoding        orotidine-5′-phosphate decarboxylase;    -   (14) Disruption of a native Y. lipolytica Leu2 gene encoding        isopropyl malate dehydrogenase;    -   (15) Disruption of a native Y. lipolytica Pox3 gene encoding        acyl-coA oxidase;    -   (16) Disruption of a native Y. lipolytica gene encoding Δ12        desaurase;    -   (17) Disruption of a native Y. lipolytica Lip1 gene encoding        lipase 1; and,    -   (18) Disruption of a native Y. lipolytica Pox2 gene encoding        acyl-CoA oxidase.

Although the Applicants demonstrate production of 5.6% DHA (wherein thecumulative total % of C22 fatty acids with respect to the total fattyacids is ˜24%) in this particular recombinant strain of Yarrowialipolytica, it is contemplated that the concentration of DHA in the hostcells could be dramatically increased via additional geneticmodifications, according to the invention herein. Furthermore, on thebasis of the teachings and results described herein, it is expected thatone skilled in the art will recognize the feasability and commercialutility created by using oleaginous yeast as a production platform forthe synthesis of a variety of ω-3 and/or ω-6 PUFAs, using the ω-6 Δ6desaturase/Δ6 elongase pathway and/or the ω-3 Δ6 desaturase/Δ6 elongasepathway and/or the ω-6 Δ9 elongase/Δ8 desaturase pathway and/or the ω-3Δ9 elongase/Δ8 desaturase pathway.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by: 1.) Sambrook,J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A LaboratoryManual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989)(Maniatis); 2.) T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions; Cold Spring Harbor Laboratory: ColdSpring Harbor, N.Y. (1984); and 3.) Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth ofmicrobial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, Eds), American Society for Microbiology: Washington,D.C. (1994)); or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, 2^(nd) ed., Sinauer Associates: Sunderland,Mass. (1989). All reagents, restriction enzymes and materials used forthe growth and maintenance of microbial cells were obtained from AldrichChemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.),GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis,Mo.), unless otherwise specified.

E. coli (XL1 -Blue) competent cells were purchased from the StratageneCompany (San Diego, Calif.). E. coli strains were typically grown at 37°C. on Luria Bertani (LB) plates.

General molecular cloning was performed according to standard methods(Sambrook et al., supra). Oligonucleotides were synthesized bySigma-Genosys (Spring, Tex.). Individual PCR amplification reactionswere carried out in a 50 μl total volume, comprising: PCR buffer(containing 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 8.75), 2 mMMgSO₄, 0.1% Triton X-100), 100 μg/mL BSA (final concentration), 200 μMeach deoxyribonucleotide triphosphate, 10 pmole of each primer and 1 μlof Pfu DNA polymerase (Stratagene, San Diego, Calif.), unless otherwisespecified. Site-directed mutagenesis was performed using Stratagene'sQuickChange™ Site-Directed Mutagenesis kit, per the manufacturers'instructions. When PCR or site-directed mutagenesis was involved insubcloning, the constructs were sequenced to confirm that no errors hadbeen introduced to the sequence. PCR products were cloned into Promega'spGEM-T-easy vector (Madison, Wis.).

DNA sequence was generated on an ABI Automatic sequencer using dyeterminator technology (U.S. Pat. No. 5,366,860; EP 272,007) using acombination of vector and insert-specific primers. Sequence editing wasperformed in Sequencher (Gene Codes Corporation, Ann Arbor, Mich.). Allsequences represent coverage at least two times in both directions.Comparisons of genetic sequences were accomplished using DNASTARsoftware (DNA Star, Iric.). Alternatively, manipulations of geneticsequences were accomplished using the suite of programs available fromthe Genetics Computer Group Inc. (Wisconsin Package Version 9.0,Genetics Computer Group (GCG), Madison, Wis.). The GCG program “Pileup”was used with the gap creation default value of 12, and the gapextension default value of 4. The GCG “Gap” or “Bestfit” programs wereused with the default gap creation penalty of 50 and the default gapextension penalty of 3. Unless otherwise stated, in all other cases GCGprogram default parameters were used.

BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J.Mol. Biol. 215:403-410 (1993) and Nucleic Acids Res. 25:3389-3402(1997)) searches were conducted to identity isolated sequences havingsimilarity to sequences contained in the BLAST “nr” database (comprisingall non-redundant GenBank CDS translations, sequences derived from the3-dimensional structure Brookhaven Protein Data Bank, the SWISS-PROTprotein sequence database, EMBL and DDBJ databases). Sequences weretranslated in all reading frames and compared for similarity to allpublicly available protein sequences contained in the “nr” database,using the BLASTX algorithm (Gish, W. and States, D. J. Nature Genetics3:266-272 (1993)) provided by the NCBI.

The results of BLAST comparisons summarizing the sequence to which aquery sequence had the most similarity are reported according to the %identity, % similarity, and Expectation value. “% Identity” is definedas the percentage of amino acids that are identical between the twoproteins. “% Similarity” is defined as the percentage of amino acidsthat are identical or conserved between the two proteins. “Expectationvalue” estimates the statistical significance of the match, specifyingthe number of matches, with a given score, that are expected in a searchof a database of this size absolutely by chance.

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “h” means hour(s), “d” means day(s), “μL” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “μmole” mean micromole(s), “g” means gram(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means basepair(s), and “kB” means kilobase(s).

Transformation and Cultivation of Yarrowia lipolytica

Yarrowia lipolytica strains ATCC #20362, #76982 and #90812 werepurchased from the American Type Culture Collection (Rockville, Md.). Y.lipolytica strains were usually grown at 28° C. on YPD agar (1% yeastextract, 2% bactopeptone, 2% glucose, 2% agar). Alternatively, “SD”media comprises: 0.67% yeast nitrogen base with ammonium sulfate,without amino acids and 2% glucose.

Transformation of Y. lipolytica was performed according to the method ofChen, D. C. et al. (Appl. Microbiol Biotechnol. 48(2):232-235 (1997)),unless otherwise noted. Briefly, Yarrowia was streaked onto a YPD plateand grown at 30° C. for approximately 18 hr. Several large loopfuls ofcells were scraped from the plate and resuspended in 1 mL oftransformation buffer containing: 2.25 mL of 50% PEG, average MW 3350;0.125 mL of2 M Li acetate, pH 6.0; 0.125 mL of2 M DTT; and 50 μg shearedsalmon sperm DNA. Then, approximately 500 ng of linearized plasmid DNAwas incubated in 100 μl of resuspended cells, and maintained at 39° C.for 1 hr with vortex mixing at 15 min intervals. The cells were platedonto selection media plates and maintained at 30° C. for 2 to 3 days.

For selection of transformants, SD medium or minimal medium (“MM”) wasgenerally used; the composition of MM is as follows: 0.17% yeastnitrogen base (DIFCO Laboratories, Detroit, Mich.) without ammoniumsulfate or amino acids, 2% glucose, 0.1% proline, pH 6.1). Supplementsof adenine, leucine, lysine and/or uracil were added as appropriate to afinal concentration of 0.01% (thereby producing “MMA”, “MMLe”, “MMLy”and “MMU” selection media, each prepared with 20 g/L agar).

Alternatively, transformants were selected on 5-fluoroorotic acid(“FOA”; also 5-fluorouracil-6-carboxylic acid monohydrate) selectionmedia, comprising: 0.17% yeast nitrogen base (DIFCO Laboratories)without ammonium sulfate or amino acids, 2% glucose, 0.1% proline, 75mg/L uracil, 75 mg/L uridine, 900 mg/L FOA (Zymo Research Corp., Orange,Calif.) and 20 g/L agar.

Finally, for the “two-stage growth conditions” designed to promoteconditions of oleaginy, High Glucose Media (“HGM”) was prepared asfollows: 14 g/L KH₂PO₄, 4 g/LK₂HPO₄, 2 g/L MgSO₄.7H₂0, 80 g/L glucose(pH 6.5). Strains were cultured under “two-stage growth conditions”according to the following protocol: first, cells were grown intriplicate in liquid MM at 30° C. with shaking at 250 rpm/min for 48hrs. The cells were collected by centrifugation and the liquidsupernatant was extracted. The pelleted cells were resuspended in HGMand grown for either 72 hrs or 96 hrs at 30° C. with shaking at 250rpm/min. The cells were again collected by centrifugation and the liquidsupernatant was extracted.

A modified media used for some of the “two-stage growth conditions” was“SD+AA” media, which consisted of the following: 6.7 g Yeast NitrogenBase without amino acids, but with ammonium sulfate, 20 g glucose, and1× amino acid mix (20 mg/mL adenine sulfate, 20 mg/mL uracil, 20 mg/mLL-tryptophan, 20 mg/mL L-histidine-HCL, 20 mg/mL L-arginine-HCL, 20mg/mL L-methionine, 30 mg/mL L-tyrosine, 30 mg/mL L-leucine, 30 mg/mLL-isoleucine, 30 mg/mL L-lysine-HCl, 50 mg/mL L-phenylalanine, 100 mg/mLL-glutamic acid, 100 mg/mL L-aspartic acid, 150 mg/mL L-valine, 200mg/mL L-threonine and 400 mg/mL L-serine).

Fatty Acid Analysis of Yarrowia lipolytica

For fatty acid analysis, cells were collected by centrifugation andlipids were extracted as described in Bligh, E. G. & Dyer, W. J. (Can.J. Biochem. Physiol. 37:911-917 (1959)). Fatty acid methyl esters wereprepared by transesterification of the lipid extract with sodiummethoxide (Roughan, G., and Nishida I. Arch Biochem Biophys.276(1):38-46 (1990)) and subsequently analyzed with a Hewlett-Packard6890 GC fitted with a 30-m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard)column. The oven temperature was from 170° C. (25 min hold) to 185° C.at 3.5° C./min.

For direct base transesterification, Yarrowia culture (3 mL) washarvested, washed once in distilled water, and dried under vacuum in aSpeed-Vac for 5-10 min. Sodium methoxide (100 μl of 1%) was added to thesample, and then the sample was vortexed and rocked for 20 min. Afteradding 3 drops of 1 M NaCl and 400 μl hexane, the sample was vortexedand spun. The upper layer was removed and analyzed by GC as describedabove.

Example 1 Identification of Promoters for High Expression in Yarrowialipolytica

Comparative studies investigating the promoter activities of the TEF,GPD, GPDIN, GPM, GPAT, FBA, FBAIN and YAT1 promoters were performed, bysynthesizing constructs comprising each promoter and the E. coli geneencoding β-glucuronidase (GUS) as a reporter gene (Jefferson, R. A.Nature. 14(342):837-838 (1989)). Then, GUS activity was measured byhistochemical and fluorometric assays (Jefferson, R. A. Plant Mol. Biol.Reporter 5:387-405 (1987)) and/or by using Real Time PCR for mRNAquantitation.

Construction of Plasmids Comprising A Chimeric Promoter::GUS::XPR Gene

Plasmid pY5-30 (FIG. 6A; SEQ ID NO:154) contained: a Yarrowia autonomousreplication sequence (ARS18); a ColE1 plasmid origin of replication; anampicillin-resistance gene (Amp^(R)), for selection in E. coli; aYarrowia LEU2 gene, for selection in Yarrowia; and a chimericTEF::GUS::XPR gene. Based on this plasmid, a series of plasmids werecreated wherein the TEF promoter was replaced with a variety of othernative Y. lipolytica promoters.

The putative promoter regions were amplified by PCR, using the primersshown below in Table 11 and either genomic Y. lipolytica DNA as templateor a fragment of genomic DNA containing an appropriate region of DNAcloned into the pGEM-T-easy vector (Promega, Madison, Wis.).

TABLE 11 Construction of Plasmids Comprising A ChimericPromoter::GUS::XPR Gene Location With Respect to Plasmid PromoterPrimers Gene RE Sites Name GPD YL211, −968 bp to the ‘ATG’ SalI andpYZGDG YL212 translation initiation site of NcoI (SEQ ID the gpd geneNOs: 225 and (SEQ ID NO: 210) 226) GPDIN YL376, YL377 −973 bp to +201 bpPstI/NcoI pDMW222 (SEQ ID NOs: around the the gpd gene (for 227 and 228)(thereby including a 146 bp promoter) intron wherein the intron is andPstI/ located at position +49 bp SalI (for to +194 bp) (SEQ ID NO:vector) 211) GPM YL203, YL204 −875 bp to the ‘ATG’ NcoI and pYZGMG (SEQID NOs: translation initiation site of SalI 229 and 230) the gpm gene(SEQ ID NO: 212) GPAT GPAT-5-1, −1130 bp to the ‘ATG’ SalI and pYGPAT-GPAT-5-2 translation initiation site of NcoI GUS (SEQ ID NOs: the gpatgene (SEQ ID 231 and 232) NO: 216) FBA ODMW314, −1001 bp to −1 bp aroundNcoI and pDMW212 YL341 the fba gene SalI (SEQ ID NOs: (SEQ ID NO: 213)233 and 234) FBAIN ODMW320, −804 bp to +169 bp NcoI and pDMW214 ODMW341around the fba gene (thereby SalI (SEQ ID NOs: including a 102 bp intron235 and 236) wherein the intron is located at position +62 bp to +165bp) (SEQ ID NO: 214) YAT1 27203-F, −778 bp to −1 bp around HindIII andpYAT-GUS 27203-R the yat1 gene SalI; also (SEQ ID NOs: (SEQ ID NO: 217)NcoI and 237 and 238) HindIII Note: The ‘A’ nucleotide of the ‘ATG’translation initiation codon was designated as +1.

The individual PCR amplification reactions for GPD, GPDIN, GPM, FBA andFBAIN were carried out in a 50 μl total volume, as described in theGeneral Methods. The thermocycler conditions were set for 35 cycles at95° C. for 1 min, 56° C. for 30 sec and 72° C. for 1 min, followed by afinal extension at 72° C. for 10 min.

The PCR amplification for the GPAT promoter was carried out in a 50 μltotal volume using a 1:1 dilution of a premixed 2×PCR solution (TaKaRaBio Inc., Otsu, Shiga, 520-2193, Japan). The final composition contained25 mM TAPS (pH 9.3), 50 mM KCl, 2 mM MgCl₂, 1 mM 2-mercaptoethanol, 200μM each deoxyribonucleotide triphosphate, 10 pmole of each primer, 50 ngtemplate and 1.25 U of TaKaRa Ex Taq™ DNA polymerase (Takara Mirus Bio,Madison, Wis.). The thermocycler conditions were set for 30 cycles at94° C. for 2.5 min, 55° C. for 30 sec and 72° C. for 2.5 min, followedby a final extension at 72° C. for 6 min.

The PCR amplification for the YAT1 promoter was carried out in acomposition comparable to that described above for GPAT. The reactionmixture was first heated to 94° C. for 150 sec. Amplification wascarried out for 30 cycles at 94° C. for 30 sec, 55° C. for 30 sec and72° C. for 1 min, followed by a final extension for 7 min at 72° C.

Each PCR product was purified using a Qiagen PCR purification kit andthen digested with restriction enzymes (according to the Table aboveusing standard conditions) and the digested products were purifiedfollowing gel electrophoresis in 1% (w/v) agarose. The digested PCRproducts (with the exception of those from YAT1) were then ligated intosimilarly digested pY5-30 vector. Ligated DNA from each reaction wasthen used to individually transform E. coli Top10, E. coli DH10B or E.coli DH5α. Transformants were selected on LB agar containing ampicillin(100 μg/mL).

YAT1 required additional manipulation prior to cloning into pY5-30.Specifically, upon digestion of the YAT1 PCR product with HindIII andSaII, a ˜600 bp fragment resulted; digestion with NcoI and HindIIIresulted in a ˜200 bp fragment. Both products were isolated andpurified. Then, plasmid pYGPAT-GUS was digested with SalI and NcoI, anda ˜9.5 kB fragment was isolated and purified. The three DNA fragmentswere ligated together to create pYAT-GUS.

Analysis of the plasmid DNA from each transformation reaction confirmedthe presence of the expected plasmid. These plasmids were designated asfollows: pYZGDG (comprising a GPD::GUS::XPR chimeric gene), pDMW222(comprising a GPDIN::GUS::XPR chimeric gene), pYZGMG (comprising aGPM::GUS::XPR chimeric gene), pYGPAT-GUS (comprising a GPAT::GUS::XPRchimeric gene), pDMW212 (comprising a FBA::GUS::XPR chimeric gene),pDMW214 (comprising a FBAIN::GUS::XPR chimeric gene) and pYAT-GUS(comprising a YAT1::GUS::XPR chimeric gene). 30 Each of the plasmidsabove, and additionally plasmid pY5-30 (comprising a TEF::GUS::XPRchimeric gene), was transformed separately into Y. lipolytica asdescribed in the General Methods. The Y. lipolytica host was either Y.lipolytica ATCC #76982 or Y. lipolytica ATCC #20362, strain Y2034 (infra[Example 13], capable of producing 10% ARA via the ω-6 Δ6 desaturase/Δ6elongase pathway). All transformed cells were plated onto minimal mediaplates lacking leucine and maintained at 30° C. for 2 to 3 days.

Comparative Analysis of Yarrowia Promoters By Histochemical Analysis ofGUS Expression

Yarrowia lipolytica ATCC #76982 strains containing plasmids pY5-30,pYZGDG, pYZGMG, pDMW212 and pDMW214 were grown from single colonies in 3mL MM at 30° C. to an OD₆₀₀˜1.0. Then, 100 μl of cells were collected bycentrifugation, resuspended in 100 μl of histochemical staining buffer,and incubated at 30° C. Staining buffer was prepared by dissolving 5 mgof 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) in 50 μl dimethylformamide, followed by the addition of 5 mL 50 mM NaPO₄, pH 7.0. Theresults of histochemical staining (FIG. 6B) showed that the TEF promoterin construct pY5-30, the GPD promoter in construct pYZGDG, the GPMpromoter in construct pYZGMG, the FBA promoter in construct pDMW212, andthe FBAIN promoter in construct pDMW214 were all active. Both the FBAand FBAIN promoters appeared to be much stronger than all the otherpromoters, with the FBAIN promoter having the strongest promoteractivity.

In a separate experiment, Y. lipolytica Y2034 strains containingplasmids pY5-30, pYGPAT-GUS, pYAT-GUS and pDMW214 were grown from singlecolonies in 5 mL SD media at 30° C. for 24 hrs to an OD₆₀₀˜8.0. Then, 1mL of cells were collected by centrifugation. The remaining cultureswere centrifuged and washed 2× with HGM, resuspended in 5 mL each of HGMand allowed to grow at 30° C. further. After 24 and 120 hrs, ˜0.25 mL ofeach culture were centrifuged to collect the cells. Cell samples wereresuspended individually in 100 μl of histochemical staining buffer(supra). Zymolase 20T (5 μl of 1 mg/mL; ICN Biomedicals, Costa Mesa,Calif.) was added to each, and the mixture incubated at 30° C.

The results of histochemical staining showed that the GPAT promoter inconstruct pYGPAT-GUS was active, as was the YAT1 promoter in constructpYAT-GUS, when grown in SD medium for 24 hrs (FIG. 6C, “24 hr in SDmedium”). Comparatively, the GPAT promoter appeared to be much strongerthan the TEF promoter and had diminished activity with respect to theFBAIN promoter. Likewise, the YAT1 promoter appeared to be stronger thanthe TEF promoter but significantly weaker than the FBAIN promoter andGPAT promoter, when cells were grown in SD medium for 24 hrs. Moreinterestingly, however, it appeared that the YAT1 promoter was strongerthan the GPAT promoter and comparable with the FBAIN promoter in cellsgrown in HGM for 24 hrs (FIG. 6C, “24 hr in HG medium”). This remainedtrue after 120 hrs in HGM (FIG. 6C, “120 hr in HG medium”). Thus, theYAT1 promoter appeared to be induced in HGM, a medium that promotesoleaginous growth conditions due to nitrogen limitation.

Comparative Analysis of Yarrowia Promoters By Fluorometric Assay of GUSExpression

GUS activity was also assayed by fluorometric determination of theproduction of 4-methylumbelliferone (4-MU) from the correspondingsubstrate β-glucuronide (Jefferson, R. A. Plant Mol. Biol. Reporter5:387-405 (1987)).

Yarrowia lipolytica ATCC #76982 strains containing plasmids pY5-30,pYZGDG, pYZGMG, pDMW212 and pDMW214 were grown from single colonies in 3mL MM (as described above) at 30° C. to an OD₆₀₀˜1.0. Then, the 3 mLcultures were each added to a 500 mL flask containing 50 mL MM and grownin a shaking incubator at 30° C. for about 24 hrs. The cells werecollected by centrifugation, resuspended in Promega Cell Lysis Bufferand lysed using the BIO 101 Biopulverizer system (Vista, Calif.). Aftercentrifugation, the supernatants were removed and kept on ice.

Similarly, Y. lipolytica strain Y2034 containing plasmids pY5-30,pYAT-GUS, pYGPAT-GUS and pDMW214 constructs, respectively, were grownfrom single colonies in 10 mL SD medium at 30° C. for 48 hrs to anOD₆₀₀˜5.0. Two mL of each culture was collected for GUS activity assays,as described below, while 5 mL of each culture was switched into HGM.

Specifically, cells from the 5 mL aliquot were collected bycentrifugation, washed once with 5 mL of HGM and resuspended in HGM. Thecultures in HGM were then grown in a shaking incubator at 30° C. for 24hrs. Two mL of each HGM culture were collected for the GUS activityassay, while the remaining culture was allowed to grow for an additional96 hrs before collecting an additional 2 mL of each culture for theassay.

Each 2 mL culture sample in SD medium was resuspended in 1 mL of 0.5×cell culture lysis reagent (Promega). Resuspended cells were mixed with0.6 mL of glass beads (0.5 mm diameter) in a 2.0 mL screw cap tube witha rubber O-ring. The cells were then homogenized in a Biospec minibeadbeater (Bartlesville, Okla.) at the highest setting for 90 sec. Thehomogenization mixtures were centrifuged for 2 min at 14,000 rpm in anEppendof centrifuge to remove cell debris and beads. The supernatant wasused for GUS assay and protein determination.

For each fluorometric assay, 100 μl of extract was added to 700 μl ofGUS assay buffer (2 mM 4-methylumbelliferyl-p-D-glucuronide (“MUG”) inextraction buffer) or 200 μl of extract was added to 800 μl of GUS assaybuffer. The mixtures were placed at 37° C. Aliquots of 100 μl were takenat 0, 30 and 60 min time points and added to 900 μl of stop buffer (1 MNa₂CO₃). Each time point was read using a CytoFluor Series 4000Fluorescence Multi-Well Plate Reader (PerSeptive Biosystems, Framingham,Mass.) set to an excitation wavelength of 360 nm and an emissionwavelength of 455 nm. Total protein concentration of each sample wasdetermined using 10 μl of extract and 200 μl of BioRad Bradford reagentor 20 μl of extract and 980 μl of BioRad Bradford reagent (Bradford, M.M. Anal. Biochem. 72:248-254 (1976)). GUS activity was expressed asnmoles of 4-MU per minute per mg of protein.

Results of these fluorometric assays designed to compare the TEF, GPD,GPM, FBA and FBAIN promoters in Y. lipolytica ATCC #76982 strains areshown in FIG. 7A. Specifically, the FBA promoter was 2.2 times strongerthan the GPD promoter in Y. lipolytica. Additionally, the GUS activityof the FBAIN promoter was about 6.6 times stronger than the GPDpromoter.

Results of these fluorometric assays designed to compare the TEF, GPAT,YAT1 and FBAIN promoters in Y. lipolytica strain Y2034 are shown in theTable below.

TABLE 12 Comparison of TEF, FBAIN, YAT1 And GPAT Promoter-Activity UnderVarious Growth Conditions Culture Promoter Conditions TEF FBAIN YAT1GPAT  48 hr, SD 0.401 43.333 0.536 5.252  24 hr, HGM 0.942 30.694 19.1542.969 120 hr HGM 0.466 17.200 13.400 3.050

Based on the data above wherein the activity of the YAT1 promoter wasquantitated based on GUS activity of cell extracts, the activity of theYAT1 promoter increased by ˜37 fold when cells were switched from SDmedium into HGM and grown for 24 hrs. After 120 hrs in HGM, the activitywas reduced somewhat but was still 25× higher than the activity in SDmedium. In contrast, the activity of the FBAIN promoter and the GPATpromoter was reduced by 30% and 40%, respectively, when switched from SDmedium into HGM for 24 hrs. The activity of the TEF promoter increasedby 2.3 fold after 24 hrs in HGM. Thus, the YAT1 promoter is inducibleunder oleaginous conditions.

Comparative Analysis of Yarrowia Promoters By Quantitative PCR Analysesof GUS Expression

The transcriptional activities of the TEF, GPD, GPDIN, FBA and FBAINpromoters were determined in Y. lipolytica containing the pY5-30,pYZGDG, pDMW222, pDMW212 and pDMW214 constructs by quantitative PCRanalyses. This required isolation of RNA and real time RT-PCR.

More specifically, Y. lipolytica ATCC #76982 strains containing pY5-30,pYZGDG, pDMW222, pDMW212 and pDMW214 were grown from single colonies in6 mL of MM in 25 mL Erlenmeyer flasks for 16 hrs at 30° C. Each of the 6mL starter cultures was then added to individual 500 mL flaskscontaining 140 mL HGM and incubated at 30° C. for 4 days. In eachinterval of 24 hrs, 1 mL of each culture was removed from each flask tomeasure the optical density, 27 mL was removed and used for afluorometric GUS assay (as described above), and two aliquots of 1.5 mLwere removed for RNA isolation. The culture for RNA isolation wascentrifuged to produce a cell pellet.

The RNA was isolated from Yarrowia strains according to the modifiedQiagen RNeasy mini protocol (Qiagen, San Diego, Calif.). Briefly, ateach time point for each sample, 340 μL of Qiagen's buffer RLT was usedto resuspend each of the two cell pellets. The buffer RLT/cellsuspension mixture from each of the two tubes was combined in a beadbeating tube (Bio101, San Diego, Calif.). About 500 μL of 0.5 mL glassbeads was added to the tube and the cells were disrupted by bead beating2 min at setting 5 (BioPulverizer, Bio101 Company, San Diego, Calif.).The disrupted cells were then pelleted by centrifugation at 14,000 rpmfor 1 min and 350 μl of the supernatent was transferred to a newmicrocentrifuge tube. Ethanol (350 μL of 70%) was added to eachhomogenized lysate. After gentle mixing, the entire sample was added toa RNeasy mini column in a 2 mL collection tube. The sample wascentrifuged for 15 sec at 10,000 rpm. Buffer RW1 (350 μL) was added tothe RNeasy mini column and the column was centrifuged for 15 sec at10,000 rpm to wash the cells. The eluate was discarded. Qiagen's DNaselstock solution (10 μL) was added to 70 μl of Buffer RDD and gentlymixed. This entire DNase solution was added to the RNeasy mini columnand incubated at room temperature for 15 min. After the incubation step,350 μL of Buffer RW1 was added to the mini column and the column wascentrifuged for 15 sec at 10,000 rpm. The column was washed twice with700 μL Buffer RW1. RNase-free water (50 μL) was added to the column. Thecolumn was centrifuged for 1 min at 10,000 rpm to elute the RNA.

A two-step RT-PCR protocol was used, wherein total Yarrowia RNA wasfirst converted to cDNA and then cDNA was analyzed using Real Time PCR.The conversion to cDNA was performed using Applied Biosystems' HighCapacity cDNA Archive Kit (PN#4322171; Foster City, Calif.) andMolecular Biology Grade water from MediaTech, Inc. (PN# 46-000-Con;Holly Hill, Fla.). Total RNA from Yarrowia (100 ng) was converted tocDNA by combining it with 10 μl of RT buffer, 4 μl of 25× dNTPs, 10 μl10× Random Hexamer primers, 5 μl Multiscribe Reverse Transcriptase and0.005 μl RNase Inhibitor, and brought to a total reaction volume of 100μl with water. The reactions were incubated in a thermocycler for 10 minat 25° C. followed by 2 hrs at 37° C. The cDNA was stored at −20° C.prior to Real Time analysis.

Real Time analysis was performed using the SYBR Green PCR Master Mixfrom Applied Biosystems (PN#4309155). The Reverse Transcription reaction(2 μl) was added to 10 μl of 2× SYBR PCR Mix, 0.2 μl of 100 μM Forwardand Reverse primers for either URA (i.e., primers YL-URA-16F andYL-URA-78R [SEQ ID NOs:239 and 240]) or GUS (i.e., primers GUS-767F andGUS-891R [SEQ ID NO:241 and 242]) and 7.2 μl water. The reactions werethermocycled for 10 min at 95° C. followed by 40 cycles of 95° C. for 5sec and 60° C. for 1 min in an ABI 7900 Sequence Detection Systeminstrument. Real time fluorescence data was collected during the 60° C.extension during each cycle.

Relative quantitation was performed using the ΔΔCT method as per UserBulletin #2: “Relative Quantitation of Gene Expression”, AppliedBiosystems, Updated October 2001. The URA gene was used fornormalization of GUS expression. In order to validate the use of URA asa normalizer gene, the PCR efficiency of GUS and URA were compared andthey were found to be 1.04 and 0.99, respectively (where 1.00 equals100% efficiency). Since the PCR efficiencies were both near 100%, theuse of URA as a normalizer for GUS expression was validated, as was theuse of the ΔΔCT method for expression quantitation. The normalizedquantity is referred to as the ΔCT.

The GUS mRNA in each different strain (i.e., Y. lipolytica ATCC #76982strains containing the pYZGDG, pDMW222, pDMW212 and pDMW214 constructs)was quantified to the mRNA level of the strain with pY5-30 (TEF::GUS).Thus, relative quantitation of expression was calculated using the mRNAlevel of the strain with TEF::GUS as the reference sample. Thenormalized value for GPD::GUS, GPDIN::GUS, FBA::GUS and FBAIN::GUS wascompared to the normalized value of the TEF::GUS reference. Thisquantity is referred to as the ΔΔCT. The ΔΔCT values were then convertedto absolute values by utilizing the formula 2^(−ΔΔCT). These valuesrefer to the fold increase in the mRNA level of GUS in the strainscomprising the chimeric GPD::GUS, GPDIN::GUS, FBA::GUS and FBAIN::GUSgenes, as compared to the chimeric TEF::GUS gene. Using thismethodology, it was possible to compare the activity of the TEF promoterto the GPD, GPDIN, FBA and FBAIN promoters.

The results of the relative quantitation of mRNA for each GUS chimericgene are shown in FIG. 7B. More specifically, the assay showed thatafter 24 hrs in HGM, the transcription activity of FBA and FBAINpromoters was about 3.3 and 6 times stronger than the TEF promoter,respectively. Similarly, the transcription activity of the GPD and GPDINpromoters is about 2 and 4.4 times stronger than the TEF promoter,respectively. While the transcription activities of the FBA::GUS,FBAIN::GUS, GPD::GUS and GPDIN::GUS gene fusion decreased over the 4 dayperiod of the experiment, the transcriptional activity of the FBAIN andGPDIN promoters was still about 3 and 2.6 times stronger than the TEFpromoter in the final day of the experiment.

Example 2 Identification of Enhancers Useful to Increase GeneTranscription in Yarrowia lipolytica

Based on the strong promoter activities of FBAIN and GPDIN (whereinactivity was greater than that of the FBA and GPD promoters,respectively) and the identification of an intron within each promoterregion, the present work was conducted to determine whether enhancerswere present in each intron.

Specifically, two chimeric promoters consisting of a GPM::FBAIN promoterfusion and a GPM::GPDIN promoter fusion were generated to driveexpression of the GUS reporter gene. The chimeric promoters (comprisedof a “component 1” and a “component 2”) are described below in Table 13.

TABLE 13 Construction of Plasmids Comprising A Chimeric Promoter WithinA Chimeric Promoter::GUS::XPR Gene Chimeric Plasmid Promoter Component 1Component 2 Name GPM::FBAIN −1 bp to +1 bp to +171 bp region pDMW224(SEQ ID NO: −843 bp of FBAIN, wherein the 219) region intron is locatedat of GPM position +62 bp to +165 bp GPM::GPDIN −1 bp to +1 bp to +198bp region pDMW225 (SEQ ID NO: −843 bp of GPDIN, wherein the 220) regionintron is located at of GPM position +49 bp to +194 bpThe chimeric promoters were positioned such that each drove expressionof the GUS reporter gene in plasmids pDMW224 and pDMW225.

The activities of the GPM::FBAIN promoter and the GPM::GPDIN promoterwere compared with the TEF, FBAIN, GPDIN and GPM promoters by comparingthe GUS activity in Y. lipolytica strains comprising pDMW224 and pDMW225relative to the GUS activity in Y. lipolytica strains comprising pY5-30,pYZGDG, PYZGMG and pDMW214 constructs based on results fromhistochemical assays (as described in Example 1). As previouslydetermined, the FBAIN promoter was the strongest promoter. However, thechimeric GPM::FBAIN promoter and the chimeric GPM::GPDIN promoter wereboth much stronger than the GPM promoter and appeared to be equivalentin activity to the GPDIN promoter. Thus, this confirmed the existence ofan enhancer in both the GPDIN promoter and the FBAIN promoter.

One skilled in the art would readily be able to construct similarchimeric promoters, using either the GPDIN intron or the FBAIN intron.

Example 3 Sulfonylurea Selection

Genetic improvement of Yarrowia has been hampered by the lack ofsuitable non-antibiotic selectable transformation markers. The presentExample describes the development of a dominant, non antibiotic markerfor Y. lipolytica based on sulfonylurea resistance that is alsogenerally applicable to industrial yeast strains that may be haploid,diploid, aneuploid or heterozygous.

Theory and Initial Sensitivity Screening

Acetohydroxyacid synthase (AHAS) is the first common enzyme in thepathway for the biosynthesis of branched-chain amino acids. It is thetarget of the sulfonylurea and imidazolinone herbicides. As such,sulfonyl urea herbicide resistance has been reported in both microbesand plants. For example, in Saccharomyces cerevisiae, the single W586Lmutation in AHAS confers resistance to sulfonylurea herbicides (Falco,S. C., et al., Dev. Ind. Microbiol. 30:187-194 (1989); Duggleby, R. G.,et. al. Eur J. Biochem. 270:2895 (2003)).

When the amino acid sequences of wild type AHAS Y. lipolytica (GenBankAccession No. XP_(—)501277) and S. cerevisiae (GenBank Accession No.P07342) enzymes were aligned, the Trp amino acid residue at position 586of the S. cerevisiae enzyme was equivalent to the Trp residue atposition 497 of the Y. lipolytica enzyme. It was therefore hypothesizedthat W497L mutation in the Y. lipolytica enzyme would likely confersulfonylurea herbicide resistance, if the wild type cells werethemselves sensitive to sulfonylurea. Using methodology well known tothose of skill in the art, it was determined that sulfonylurea(chlorimuron ethyl) at a concentration of 100 μg/mL in minimal mediumwas sufficient to inhibit growth of wild type Y. lipolytica strains ATCC#20362 and ATCC #90812.

Synthesis of A Mutant W497L AHAS Gene

The Y. lipolytica AHAS gene containing the W497L mutation (SEQ IDNO:243) was created from genomic DNA in a two-step reaction. First, the5′ portion of the AHAS gene was amplified from genomic DNA using PfuUltra™ High-Fidelity DNA Polymerase (Stratagene, Catalog #600380) andprimers 410 and 411 [SEQ ID NOs:244 and 245]; the 3′ portion of the genewas amplified similarly using primers 412 and 413 [SEQ ID NOs:246 and237]. The two pairs of primers were overlapping such that theoverlapping region contained the W497L mutation (wherein the mutationwas a ‘CT’ change to ‘TG’).

The 5′ and 3′ PCR products of the correct size were gel purified andused as the template for the second round of PCR, wherein the entiremutant gene was amplified using primers 414 and 415 (SEQ ID NOs:248 and249) and a mixture of the products from the two primary PCR reactions.This mutant gene carried its own native promoter and terminatorsequences. The second round PCR product of the correct size was gelpurified and cloned by an in-fusion technique into the vector backboneof plasmid pY35 [containing a chimeric TEF::Fusarium moniliforme Δ12desaturase (Fm2) gene, the E. coli origin of replication, a bacterialampicillin resistance gene, the Yarrowia Leu 2 gene and the Yarrowiaautonomous replication sequence (ARS); see WO 2005/047485 for additionaldetails], following its digestion with enzymes SaII/BsiWI. The in-fusionreaction mixture was transformed into TOP10 competent cells (Invitrogen,Catalog #C4040-10). After one day selection on LB/Amp plates, eight (8)colonies were analyzed by DNA miniprep. Seven clones were confirmed tobe correct by restriction digest. One of them that contained thesulfonylurea resistance gene as well as the LEU gene was designated“pY57” (or “pY57:YI.AHAS.w497I”; FIG. 3B).

Wild type Y. lipolytica strains ATCC #90812 and #20362 were transformedwith pY57 and ‘empty’ LEU by a standard Lithium Acetate method.Transformation controls comprising ‘No-DNA’ were also utilized.Transformants were plated onto either MM or MM+sulfonylurea (SU; 100μg/mL) agar plates and the presence or absence of colonies was evaluatedfollowing four days of growth.

TABLE 14 AHAS Selection In Yarrowia lipolytica ATCC #90812 ATCC #20362MM + SU MM + SU Plasmid MM (100 μg/mL) MM (100 μg/mL) pY57 coloniescolonies colonies colonies Leu vector colonies No colonies colonies Nocolonies control No DNA No colonies No colonies No colonies No coloniescontrol

Based on the results shown above, AHAS W497L was a good non-antibioticselection marker in both Y. lipolytica ATCC #90812 and #20362.Subsequently, Applicants used a sulfonylurea concentration of 150 μg/mL.This new marker is advantageous for transforming Y. lipolytica since itdoes not rely on a foreign gene but on a mutant native gene and itneither requires auxotrophy nor results in auxotrophy. The herbicide isnon-toxic to humans and animals.

It is expected that this selection method will be generally applicableto other industrial yeast strains that may be haploid, diploid,aneuploid or heterozygous, if mutant AHAS enzymes were created in amanner analogous to that described herein.

Example 4 Synthesis and Functional Expression of A Codon-Optimized Δ4Desaturase Gene in Yarrowia lipolytica

The codon usage of the Δ4 desaturase gene of Thraustochytrium aureum(GenBank Accession No. AAN75707) was optimized for expression in Y.lipolytica, in a manner similar to that described in WO 2004/101753.Specificially, according to the Yarrowia codon usage pattern, theconsensus sequence around the ATG translation initiation codon, and thegeneral rules of RNA stability (Guhaniyogi, G. and J. Brewer, Gene265(1-2):11-23 (2001)), a codon-optimized Δ4 desaturase gene wasdesigned (SEQ ID NOs:106 and 107), based on the DNA sequence of theThraustochytrium aureum gene (SEQ ID NO:104). In addition tomodification of the translation initiation site, 170 bp of the 1545 bpcoding region were modified (11%), and 166 codons were optimized. Inorder to modify the translation initiation site, the second amino acid(T) of SEQ ID NO:105 (wild type) was not included in the codon-optimizedΔ4 desaturase gene (SEQ ID NO:107).

In Vitro Synthesis of A Codon-Optimized Δ4 Desaturase Gene

The codon-optimized Δ4 desaturase gene was synthesized as follows.First, sixteen pairs of oligonucleotides were designed to extend theentire length of the codon-optimized coding region of theThraustochytrium aureum Δ4 desaturase gene (e.g., D4-1A, D4-1B, D4-D2A,D4-2B, D4-3A, D4-3B, D4-4A, D4-4B, D4-5A, D4-5B, D4-6A, D4-6B, D4-7A,D4-7B, D4-8A, D4-8B, D4-9A, D4-9B, D4-10A, D4-10B, D4-11A, D4-11B,D4-12A, D4-12B, D4-13A, D4-13B, D4-14A, D4-14B, D4-15A, D4-15B, D4-16Aand D4-16B, corresponding to SEQ ID NOs:250-281). Each pair of sense (A)and anti-sense (B) oligonucleotides were complementary, with theexception of a 4 bp overhang at each 5′-end. Additionally, primer D4-1F(SEQ ID NO:282) introduced a NcoI site; primers D4-4R (SEQ ID NO:283)and D4-5F (SEQ ID NO:284) introduced a BamHI site; primers D4-8R (SEQ IDNO:285) and D4-9F (SEQ ID NO:286) introduced a HindIII site that changedthe #274 amino acid of Phe to Leu; primers D4-12R (SEQ ID NO:287) andD4-13 (SEQ ID NO:288) introduced an ApaLI site; and primer D4-16R (SEQID NO:289) introduced a NotI site for subsequent subcloning. The aminoacid change at position #274, introduced by primers D4-8R and D4-9F wassubsequently corrected following whole gene assembly.

Each oligonucleotide (100 ng) was phosphorylated at 37° C. for 1 hr in avolume of 20 μl containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mMDTT, 0.5 mM spermidine, 0.5 mM ATP and 10 U of T4 polynucleotide kinase.Each pair of sense and antisense oligonucleotides was mixed and annealedin a thermocycler using the following parameters: 95° C. (2 min), 85° C.(2 min), 65° C. (15 min), 37° C. (15 min), 24° C. (15 min) and 4° C. (15min). Thus, D4-1A (SEQ ID NO:250) was annealed to D4-1B (SEQ ID NO:251)to produce the double-stranded product “D4-1AB”. Similarly, D4-2A (SEQID NO:252) was annealed to D4-2B (SEQ ID NO:253) to produce thedouble-stranded product “D4-2AB”, etc.

Four separate pools of annealed, double-stranded oligonucleotides werethen ligated together, as shown below: Pool 1 (comprising D4-1AB,D4-2AB, D4-3AB and D4-4AB); Pool 2 (comprising D4-5AB, D4-6AB, D4-7ABand D4-8AB); Pool 3 (comprising D4-9AB, D4-10AB, D4-11AB and D4-12AB);and, Pool 4 (comprising D4-13AB, D4-14AB, D4-15AB and D4-16AB). Eachpool of annealed oligonucleotides was mixed in a volume of 20 μl with 10U of T4 DNA ligase and the ligation reaction was incubated overnight at16° C.

The product of each ligation reaction was then used as template toamplify the designed DNA fragment by PCR. Specifically, using theligated “Pool 1” mixture (i.e., D4-1AB, D4-2AB, D4-3AB and D4-4AB) astemplate, and oligonucleotides D4-1F and D4-4R (SEQ ID NOs:282 and 283)as primers, the first portion of the codon-optimized Δ4 desaturase genewas amplified by PCR. The PCR amplification was carried out in a 50 μltotal volume, as described in the General Methods. Amplification wascarried out as follows: initial denaturation at 95° C. for 3 min,followed by 35 cycles of the following: 95° C. for 1 min, 56° C. for 30sec, 72° C. for 40 sec. A final extension cycle of 72° C. for 10 min wascarried out, followed by reaction termination at 4° C. The 433 bp PCRfragment was subcloned into the pGEM-T easy vector (Promega) to generatepT4(1-4).

Using the ligated “Pool 2” mixture (i.e., D4-5AB, D4-6AB, D4-7AB andD4-8AB) as the template, and oligonucleotides D4-5F and D4-8R (SEQ IDNOs:284 and 285) as primers, the second portion of the codon-optimizedΔ4 desaturase gene was amplified similarly by PCR and cloned intopGEM-T-easy vector to generate pT4(5-8).

Using the ligated “Pool 3” mixture (i.e., D4-9AB, D4-10AB, D4-11AB andD4-12AB) as the template, and oligonucleotides D4-9F and D4-12R (SEQ IDNOs:286 and 287) as primers, the third portion of the codon-optimized Δ4desaturase gene was amplified similarly by PCR and cloned intopGEM-T-easy vector to generate pT4(9-12).

Using the ligated “Pool 4” mixture (i.e., D4-13AB, D4-14AB, D4-15AB andD4-16AB) as the template, and oligonucleotides D4-13F and D4-16R (SEQ IDNOs:288 and 289) as primers, the fourth portion of the codon-optimizedΔ4 desaturase gene was amplified similarly by PCR and cloned intopGEM-T-easy vector to generate pT4(13-16).

E. coli was transformed separately with pT4(1-4), pT4(5-8), pT4(9-12)and pT4(13-16) and the plasmid DNA was isolated fromampicillin-resistant transformants. Plasmid DNA was purified anddigested with the appropriate restriction endonucleases to liberate the433 bp NcoI/BamHI fragment of pT4(1-4), the 383 bp BamHI/HindIIIfragment of p4(5-8), the 436 bp HindIII/ApaLI fragment of p4(9-12), andthe 381 bp ApaLI/NotI fragment of p4(13-16). These four fragments werethen combined and directionally ligated together with Nco1/Not1 digestedpZUF17 (SEQ ID NO:162; FIG. 9B) to generate pZUF4 (SEQ ID NO:163).

The #274 amino acid of the synthetic Δ4 desaturase gene (“D4S”) in pZUF4was originally changed from Phe to Leu because of the convenience ofcloning. The #274 amino acid of Leu was corrected to Phe bysite-directed mutagenesis using pZUF4 as template and oligonucleotidesYL251 and YL252 as primers (SEQ ID NOs:290 and 291). Thus, the resultingplasmid contained the correct synthetic amino acid sequence of Δ4desaturase gene (SEQ ID NO:106) and was designated as pZUF4S (SEQ IDNO:164; FIG. 9C).

Expression of the Codon-Optimized Δ4 Desaturase Gene in Y. lipolytica

Construct pZUF4S, an auto-replication plasmid comprising a chimericFBAIN::D4S::Pex20 gene, was transformed into Yarrowia lipolytica strainY20362U (an autonomous Ura- mutant of ATCC #20362, generated byselecting for FOA resistance) as described in the General Methods. Thetransformant cells were plated onto MM selection media plates andmaintained at 30° C. for 2 to 3 days. Transformants (3) grown on the MMplates were picked and re-streaked onto fresh MM plates. Once grown,these strains were individually inoculated into 3 mL liquid MM suppliedwith 10 μg DPA at 30° C. and shaken at 250 rpm/min for 2 days. The cellswere collected by centrifugation, lipids were extracted, and fatty acidmethyl esters were prepared by trans-esterification, and subsequentlyanalyzed with a Hewlett-Packard 6890 GC.

The GC results showed that there were about 2% DHA produced in thesethree transformants. The “percent (%) substrate conversion” of thecodon-optimized gene was determined to be about 20%.

Example 5 Synthesis and Functional Expression of A Codon-OptimizedC_(20/22) Elongase Gene in Yarrowia lipolytica

The codon usage of the C_(20/22) elongase gene of Ostreococcus tauti(GenBank Accession No. AY591336; “OtElo2”) was optimized for expressionin Y. lipolytica, in a manner similar to that described in WO2004/101753 and Example 4 (supra). Specifically, a codon-optimizedOtElo2 elongase gene (designated “OtE2S”, SEQ ID NO:102) was designed,based on the published sequence of Ostreococcus tauri (GenBank AccessionNo. AY591336, SEQ ID NO:100), according to the Yarrowia codon usagepattern (WO 2004/101753), the consensus sequence around the ‘ATG’translation initiation codon, and the general rules of RNA stability(Guhaniyogi, G. and J. Brewer, Gene 265(1-2):11-23 (2001)). In additionto the modification of the translation initiation site, 160 bp of the903 bp coding region were modified (17.7%) and 147 codons were optimized(49%). None of the modifications in the codon-optimized gene changed theamino acid sequence of the encoded protein (SEQ ID NO:101), except thatthe second amino acid was changed from ‘S’ to ‘A’ in order to add theNcoI site around the translation initiation codon within thecodon-optimized ORF (SEQ ID NOs:102 and 103). The designed OtE2S genewas synthesized by GenScript Corporation (Piscataway, N.J.) and clonedinto pUC57 (GenBank Accession No. Y14837) to generate pOtE2S.

Expression of The Codon-Optimized OtE2S Gene in Y. lipolytica

The NcoI/NotI fragment of pOtE2S was isolated and ligated together withNco1/Not1 digested pZUF17 (SEQ ID NO:162; FIG. 9B) to generate pFOE2S(FIG. 9D). Construct pFOE2S, an auto-replication plasmid comprising achimeric FBAIN::OtE2S::Pex20 gene, was transformed into Yarrowialipolytica strain Y20362U (an autonomous Ura- mutant of ATCC #20362,selected under FOA screening) as described in the General Methods. Thetransformant cells were plated onto MM selection media plates andmaintained at 30° C. for 2 to 3 days. Transformants (3) of Y20362U withpFOE2S grown on MM plates were picked and re-streaked onto fresh MMplates. Once grown, these strains were individually inoculated into 3 mLliquid MM supplied with 10 μg EPA at 30° C. and shaken at 250 rpm/minfor 2 days. The cells were collected by centrifugation, lipids wereextracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

The GC results showed that there were about 1% to 1.3% DPA, and about0.5% EPA of total lipids in these three transformants. The EPA to DPA“percent (%) substrate conversion” of the codon-optimized OtE2S gene inthese transformants was determined to be 67%.

Example 6 The ω-6 Δ6 Desaturase/Δ6 Elongase Pathway: Generation ofStrain Y3000 to Produce Greater than 5% DHA of Total Lipids

The present Example describes the construction of strain Y3000, derivedfrom Yarrowia lipolytica ATCC #20362, capable of producing 5.6% DHArelative to the total lipids (FIG. 5). This strain was engineered toexpress the ω-6 Δ6 desaturase/Δ6 elongase pathway.

The development of strain Y3000 required the construction of strain M4(producing 8% DGLA), strain Y2047 (producing 11% ARA), strain Y2048(producing 11% EPA), strain Y2060 (producing 13% EPA), strain Y2072(producing 15% EPA), strain Y2072U3 (producing 16% EPA), strain Y2098(producing 22% EPA) and strain Y2098U (producing 21% EPA).

Generation of M4 Strain to Produce about 8% DGLA of Total Lipids

Construct pKUNF12T6E (FIG. 8A; SEQ ID NO:156) was generated to integratefour chimeric genes (comprising a Δ12 desaturase, a Δ6 desaturase andtwo C_(18/20) elongases) into the Ura3 loci of wild type Yarrowia strainATCC #20362, to thereby enable production of DGLA. The pKUNF12T6Eplasmid contained the following components:

TABLE 15 Description of Plasmid pKUNF12T6E (SEQ ID NO: 156) RE Sites AndNucleotides Within SEQ ID Description Of Fragment And Chimeric Gene NO:156 Components AscI/BsiWI 784 bp 5′ part of Yarrowia Ura3 gene (GenBank(9420-8629) Accession No. AJ306421) SphI/PacI 516 bp 3′ part of YarrowiaUra3 gene (GenBank (12128-1) Accession No. AJ306421) SwaI/BsiWIFBAIN::EL1S::Pex20, comprising: (6380-8629) FBAIN: FBAIN promoter (SEQID NO: 214) EL1S: codon-optimized elongase 1 gene (SEQ ID NO: 24),derived from Mortierella alpina (GenBank Accession No. AX464731) Pex20:Pex20 terminator sequence from Yarrowia Pex20 gene (GenBank AccessionNo. AF054613) BglII/SwaI TEF::Δ6S::Lip1, comprising: (4221-6380) TEF:TEF promoter (GenBank Accession No. AF054508) Δ6S: codon-optimized Δ6desaturase gene (SEQ ID NO: 3), derived from Mortierella alpina (GenBankAccession No. AF465281) Lip1: Lip1 terminator sequence from YarrowiaLip1 gene (GenBank Accession No. Z50020) PmeI/ClaI FBA::F.Δ12::Lip2,comprising: (4207-1459) FBA: FBA promoter (SEQ ID NO: 213) F.Δ12:Fusarium moniliforme Δ12 desaturase gene (SEQ ID NO: 32) Lip2: Lip2terminator sequence from Yarrowia Lip2 gene (GenBank Accession No.AJ012632) ClaI/PacI TEF::EL2S::XPR, comprising: (1459-1) TEF: TEFpromoter (GenBank Accession No. AF054508) EL2S: codon-optimized elongasegene (SEQ ID NO: 27), derived from Thraustochytrium aureum (U.S. Pat.No. 6,677,145) XPR: ~100 bp of the 3′ region of the Yarrowia Xpr gene(GenBank Accession No. M17741)

The pKUNF12T6E plasmid was digested with AscI/SphI, and then used fortransformation of wild type Y. lipolytica ATCC #20362 according to theGeneral Methods. The transformant cells were plated onto FOA selectionmedia plates and maintained at 30° C. for 2 to 3 days. The FOA resistantcolonies were picked and streaked onto MM and MMU selection plates. Thecolonies that could grow on MMU plates but not on MM plates wereselected as Ura- strains. Single colonies of Ura- strains were theninoculated into liquid MMU at 30° C. and shaken at 250 rpm/min for 2days. The cells were collected by centrifugation, lipids were extracted,and fatty acid methyl esters were prepared by trans-esterification, andsubsequently analyzed with a Hewlett-Packard 6890 GC.

GC analyses showed the presence of DGLA in the transformants containingthe 4 chimeric genes of pKUNF12T6E, but not in the wild type Yarrowiacontrol strain. Most of the selected 32 Ura⁻ strains produced about 6%DGLA of total lipids. There were 2 strains (i.e., strains M4 and 13-8)that produced about 8% DGLA of total lipids.

Generation of Y2047 Strain to Produce about 10% ARA of Total Lipids

Construct pDMW271 (FIG. 8B; SEQ ID NO:157) was generated to integratethree Δ5 chimeric genes into the Leu2 gene of Yarrowia strain M4.Plasmid pDMW271 contained the following components, as described inTable 16:

TABLE 16 Description of Plasmid pDMW271 (SEQ ID NO: 157) RE Sites AndNucleotides Within SEQ ID Description Of Fragment And Chimeric Gene NO:157 Components AscI/BsiWI 788 bp 5′ part of Yarrowia Leu2 gene (GenBank(5520-6315) Accession No. AF260230) SphI/PacI 703 bp 3′ part of YarrowiaLeu2 gene (GenBank (2820-2109) Accession No. AF260230) SwaI/BsiWIFBAIN::MAΔ5::Pex20, comprising: (8960-6315) FBAIN: FBAIN promoter (SEQID NO: 214) MAΔ5: Mortierella alpina Δ5 desaturase gene (SEQ ID NO: 6)(GenBank Accession No. AF067654) Pex20: Pex20 terminator sequence ofYarrowia Pex20 gene (GenBank Accession No. AF054613) SwaI/ClaITEF::MAΔ5::Lip1, comprising: (8960-11055) TEF: TEF promoter (GenBankAccession No. AF054508) MAΔ5: SEQ ID NO: 6 (supra) Lip1: Lip1 terminatorsequence of Yarrowia Lip1 gene (GenBank Accession No. Z50020) PmeI/ClaIYarrowia Ura3 gene (GenBank Accession No. (12690-11055) AJ306421)ClaI/PacI TEF::HΔ5S::Pex16, comprising: (1-2109) TEF: TEF promoter(GenBank Accession No. AF054508) HΔ5S: codon-optimized Δ5 desaturasegene (SEQ ID NO: 13), derived from Homo sapiens (GenBank Accession No.NP_037534) Pex16: Pex16 terminator sequence of Yarrowia Pex16 gene(GenBank Accession No. U75433)

Plasmid pDMW271 was digested with AscI/SphI, and then used to transformstrain M4 according to the General Methods. Following transformation,the cells were plated onto MMLe plates and maintained at 30° C. for 2 to3 days. The individual colonies grown on MMLe plates were picked andstreaked onto MM and MMLe plates. Those colonies that could grow on MMLeplates but not on MM plates were selected as Leu2⁻ strains. Singlecolonies of Leu2⁻ strains were then inoculated into liquid MMLe media at30° C. and shaken at 250 rpm/min for 2 days. The cells were collected bycentrifugation, lipids were extracted, and fatty acid methyl esters wereprepared by trans-esterification, and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed the presence of ARA in pDMW271 transformants, but notin the parental M4 strain. Specifically, among the 48 selected Leu2⁻transformants with pDMW271, there were 35 strains that produced lessthan 5% ARA of total lipids, 12 strains that produced 6-8% ARA, and 1strain that produced about 11% ARA of total lipids in the engineeredYarrowia. The strain that produced 11% ARA was named “Y2047”.

Generation of Y2048 Strain to Produce about 11% EPA of Total Lipids

Construct pZP3L37 (FIG. 8C; SEQ ID NO:158) was created to integratethree synthetic Δ17 desaturase chimeric genes into the acyl-CoA oxidase3 gene of the Y2047 strain. The plasmid pZP3L37 contained the followingcomponents:

TABLE 17 Description of Plasmid pZP3L37 (SEQ ID NO: 158) RE Sites AndNucleotides Within SEQ ID Description Of Fragment And Chimeric Gene NO:158 Components AscI/BsiWI 763 bp 5′ part of Yarrowia Pox3 gene (GenBank(6813-6043) Accession No. AJ001301) SphI/PacI 818 bp 3′ part of YarrowiaPox3 gene (GenBank (9521-10345) Accession No. AJ001301) ClaI/BsiWITEF::Δ17S::Pex20, comprising: (4233-6043) TEF: TEF promoter (GenBankAccession No. AF054508) Δ17S: codon-optimized Δ17 desaturase gene (SEQID NO: 21), derived from S. diclina (US 2003/0196217 A1) Pex20: Pex20terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613) ClaI/PmeI FBAIN::Δ17S::Lip2, comprising: (4233-1811) FBAIN:FBAIN promoter (SEQ ID NO: 214) Δ17S: SEQ ID NO: 21 (supra) Lip2: Lip2terminator sequence of Yarrowia Lip2 gene (GenBank Accession No.AJ012632) PmeI/SwaI Yarrowia Leu2 gene (GenBank Accession No. (1811-1)AF260230) PacI/SwaI FBAINm::Δ17S::Pex16, comprising: (10345-1) FBAINm:FBAINm promoter (SEQ ID NO: 215) Δ17S: SEQ ID NO: 21 (supra) Pex16:Pex16 terminator sequence of Yarrowia Pex16 gene (GenBank Accession No.U75433)

Plasmid pZP3L37 was digested with AscI/SphI, and then used to transformstrain Y2047 according to the General Methods. Following transformation,the cells were plated onto MM plates and maintained at 30° C. for 2 to 3days. A total of 96 transformants grown on the MM plates were picked andre-streaked onto fresh MM plates. Once grown, these strains wereindividually inoculated into liquid MM at 30° C. and shaken at 250rpm/min for 2 days. The cells were collected by centrifugation, lipidswere extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed the presence of EPA in most of the transformants withpZP3L37, but not in the parental Y2047 strain. Among the 96 selectedtransformants with pZP3L37, there were 20 strains that produced lessthan 2% EPA, 23 strains that produced 2-3% EPA, 5 strains that produced3-4% EPA, and 2 strains (i.e., strain #71 and strain #94) that producedabout 6% EPA of total lipids in the engineered Yarrowia. Strain #71(which produced 6% EPA) was further analyzed using “two-stage growthconditions”, as described in the General Methods (i.e., 48 hrs MM, 72hrs HGM). GC analyses showed that strain #71 produced about 11% EPA oftotal lipids. The strain was designated as “Y2048”.

Generation of Y2060 Strain to Produce about 13% EPA of Total Lipids withUra- Phenotype

In order to disrupt the Ura3 gene in strain Y2048, construct pZKUT16(FIG. 8D; SEQ ID NO:159) was created to integrate a TEF::rELO2S::Pex20chimeric gene into the Ura3 gene of strain Y2048. rELO2S is acodon-optimized rELO gene encoding a rat hepatic enzyme that elongates16:0 to 18:0 (i.e., a C_(16/18)elongase). The plasmid pZKUT16 containedthe following components:

TABLE 18 Description of Plasmid pZKUT16 (SEQ ID NO: 159) RE Sites AndNucleotides Within SEQ ID Description Of Fragment And Chimeric Gene NO:159 Components BsiWI/PacI 721 bp 5′ part of Yarrowia Ura3 gene (GenBank(1-721) Accession No. AJ306421) SalI/ClaI 724 bp 3′ part of YarrowiaUra3 gene (GenBank (3565-4289) Accession No. AJ306421) ClaI/BsiWITEF::rELO2S::Pex20, comprising: (4289-1) TEF: TEF promoter (GenBankAccession No. AF054508) rELO2S: codon-optimized rELO2 elongase gene (SEQID NO: 85), derived from rat (GenBank Accession No. AB071986) Pex20:Pex20 terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613)

Specifically, plasmid pZKUT16 was digested with SalI/PacI, and then usedto transform strain Y2048 according to the General Methods. Followingtransformation, cells were plated onto MM+5-FOA selection plates andmaintained at 30° C. for 2 to 3 days.

A total of 40 transformants grown on MM+5-FOA plates were picked andre-streaked onto MM plates and MM+5-FOA plates, separately. Thosestrains that could grow on MM+5-FOA plates, but not on MM plates, wereselected as Ura- strains. Each of these 40 Ura- strains wereindividually inoculated into liquid MMU and grown at 30° C. with shakingat 250 rpm/min for 2 days. The cells were collected by centrifugation,lipids were extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed that there were 14 strains that produced less than 5%EPA, 9 strains that produced 5-5.9% EPA, 15 strains that produced 6-6.9%EPA, and 7 strains that produced 7-8% EPA of total lipids after two daygrowth in MMU media. The strains that produced 7-8% EPA were furtheranalyzed using two-stage growth conditions, as described in the GeneralMethods (i.e., 48 hrs MM, 96 hrs HGM). GC analyses showed that all ofthese strains produced more than 10% EPA; and, one of them producedabout 13% EPA of the total lipids. That strain was designated as strain“Y2060”.

Generation of Y2072 Strain to Produce about 15% EPA of Total Lipids

Construct pKO2UM25E (FIG. 8E; SEQ ID NO:160) was created to integrate acluster of three chimeric genes (comprising a C_(18/20) elongase, a Δ12desaturase and a Δ5 desaturase) and a Ura3 gene into the native YarrowiaΔ12 desaturase gene site of strain Y2060. Plasmid pKO2UM25E containedthe following components:

TABLE 19 Description of Plasmid pKO2UM25E (SEQ ID NO: 160) RE Sites AndNucleotides Within SEQ ID Description Of Fragment And Chimeric Gene NO:160 Components HindIII/AscI 728 bp 5′ part of Yarrowia Δ12 desaturasegene (SEQ ID (1-728) NO: 28) SphI/EcoRI 556 bp 3′ part of Yarrowia Δ12desaturase gene (SEQ ID (3436-3992) NO: 28) BsiWI/HindIIIGPAT::EL1S::XPR, comprising: (10437-1) GPAT: GPAT promoter (SEQ ID NO:216) EL1S: codon-optimized elongase 1 gene (SEQ ID NO: 24), derived fromMortierella alpina (GenBank Accession No. AX464731) XPR: ~100 bp of the3′ region of the Yarrowia Xpr gene (GenBank Accession No. M17741)BglII/BsiWI FBAIN::M.Δ12::Pex20, comprising: (7920-10437) FBAIN: FBAINpromoter (SEQ ID NO: 214) M.Δ12: Mortierella isabellina Δ12 desaturasegene (GenBank Accession No. AF417245; SEQ ID NO: 30) Pex20: Pex20terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613) SalI/PacI Yarrowia Ura3 gene (Gene Bank Accession No.(6046-7544) AJ306421) EcoRI/SalI TEF::I.Δ5S::Pex20, comprising:(3992-6046) TEF: TEF promoter (GenBank Accession No. AF054508) I.Δ5S:codon-optimized Δ5 desaturase gene (SEQ ID NO: 10), derived fromIsochrysis galbana (WO 2002/ 081668) Pex20: Pex20 terminator sequence ofYarrowia Pex20 gene (GenBank Accession No. AF054613)

Specifically, plasmid pKO2UM25E was digested with SphI/AscI, and thenused to transform Y2060 according to the General Methods. Followingtransformation, cells were plated onto MM plates and maintained at 30°C. for 2 to 3 days.

A total of 63 transformants grown on MM plates were picked andre-streaked onto fresh MM plates. Once grown, these strains wereindividually inoculated into liquid MM at 30° C. and cultured withshaking at 250 rpm/min for 2 days. The cells were collected bycentrifugation, lipids were extracted, and fatty acid methyl esters wereprepared by trans-esterification, and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed the presence of EPA in almost all transformants withpKO2UM25E after one-day growth in MM media. Among the 63 selectedtransformants, there were 26 strains that produced 6-8.9% EPA and 46strains that produced more than 9% EPA. The strains that produced morethan 9% EPA were selected for further analysis using two-stage growthconditions, as described in the General Methods (i.e., 48 hrs MM, 96 hrsHGM). GC analyses showed that 45 out of the 46 selected strains produced11-14.5% EPA while culture #2 produced 15.1% EPA of total lipids afterthe two-stage growth. This strain (i.e., #2) was designated as strain“Y2072”.

Generation of Y2072U3 and Y2072U4 Strains to Produce about 15-16% EPA ofTotal Lipids with Ura- Phenotype

The construct pZKUT16 (FIG. 8D, SEQ ID NO:159; supra) was used tointegrate a TEF::rELO2S::Pex20 chimeric gene into the Ura3 gene ofstrain Y2072. Specifically, SalI/PacI-digested plasmid pZKUT16 was usedto transform strain Y2072 according to the General Methods. Followingtransformation, cells were plated onto MM+5-FOA selection plates andmaintained at 30° C. for 3 to 4 days.

A total of 24 transformants grown on MM+5-FOA plates were picked andre-streaked onto MM plates and MM+5-FOA plates, separately. The strainsthat could grow on MM+5-FOA plates, but not on MM plates, were selectedas Ura- strains. These 24 Ura- strains were individually inoculated intoliquid MMU at 30° C. and cultured with shaking at 250 rpm/min for 2days. The cells were collected by centrifugation, lipids were extracted,and fatty acid methyl esters were prepared by trans-esterification, andsubsequently analyzed with a Hewlett-Packard 6890 GC.

GC analyses showed that there were 14 strains that produced less than8.9% EPA, 8 strains that produced 9-9.9% EPA, and 1 strain (i.e., #12)that produced 10.1% EPA of total lipids after two day growth in MMUmedia. Strains #12 (10.1% EPA) and #11 (9.6% EPA) were further analyzedusing the two-stage growth procedure (i.e., 48 hrs MM, 96 hrs HGM). GCanalyses showed that strain #12 produced about 15% EPA and this strainwas designated as strain “Y2072U3”. In contrast, strain #11 producedabout 16% EPA and this strain was designated as strain “Y2072U4”.

Generation of Y2096, Y2097, Y2098, Y2105 and Y2106 Strains to Produce23-28% EPA of Total Lipids

Construct pDMW303 (FIG. 9A, SEQ ID NO:161) was created to integrate acluster of four chimeric genes (comprising a C_(18/20) elongase, a Δ6desaturase, a Δ5 desaturase and a Al 2 desaturase) and a Ura3 gene intothe Yarrowia lipase1 gene site of strain Y2072U3. Plasmid pDMW303contained the following components:

TABLE 20 Description of Plasmid pDMW303 (SEQ ID NO: 161) RE Sites AndNucleotides Within SEQ ID Description Of Fragment And Chimeric Gene NO:161 Components BsiWI/AscI 819 bp 5′ part of Yarrowia lipase1 gene(GenBank (1-819) Accession No. Z50020) SphI/PacI 769 bp 3′ part ofYarrowia lipase1 gene (GenBank (35278-4297) Accession No. Z50020)SwaI/BsiWI GPAT::HΔ5S::Pex20, comprising: (13300-1) GPAT: GPAT promoter(SEQ ID NO: 216) HΔ5S: codon-optimized Δ5 desaturase gene (SEQ ID NO:13), derived from Homo sapiens (GenBank Accession No. NP_037534) Pex20:Pex20 terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613) BglII/SwaI FBAIN::D65::Lip1, comprising: (10602-13300) FBAIN:FBAIN promoter (SEQ ID NO: 214) Δ6S: codon-optimized Δ6 desaturase gene(SEQ ID NO: 3), derived from Mortierella alpina (GenBank Accession No.AF465281) Lip1: Lip1 terminator sequence from Yarrowia Lip1 gene(GenBank Accession No. Z50020) ClaI/PmeI GPDIN::EL1S::Lip2, comprising:(8081-10558) GPDIN: GPDIN promoter (SEQ ID NO: 211) EL1S:codon-optimized elongase 1 gene (SEQ ID NO: 24), derived fromMortierella alpina (GenBank Accession No. AX464731) Lip2: Lip2terminator of Yarrowia lipase2 gene (GenBank Accession No. AJ012632)EcoRI/ClaI Yarrowia Ura 3 gene (GenBank Accession No. (6453-8081)AJ306421) PacI/EcoRI TEF::F.Δ12::Pex16, comprising: (4297-6453) TEF: TEFpromoter (GenBank Accession No. AF054508) F.Δ12: Fusarium moniliformeΔ12 desaturase gene (SEQ ID NO: 32) Pex16: Pex16 terminator of YarrowiaPex16 gene (GenBank Accession No. U75433)

Specifically, SphI/AscI-digested plasmid was transformed into strainY2072U3 according to the General Methods. Following transformation,cells were plated onto MM plates and maintained at 30° C. for 3 to 4days.

A total of 48 transformants grown on MM plates were picked andre-streaked onto fresh MM plates. Once grown, these strains wereindividually inoculated into liquid MM at 30° C. and grown with shakingat 250 rpm/min for 2 days. The cells were collected by centrifugation,lipids were extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed that EPA was produced in almost all transformants ofY2072U3 with pDMW303 after two days growth in MM. Among the 48 selectedtransformants, there were 35 strains that produced less than 13.9% EPA,8 strains that produced 14-16.9% EPA, and 4 strains that produced17-18.3% EPA of total lipids.

Those strains producing more than 14% EPA of total lipids (i.e., after 2days in MM) were selected for further analysis using the two-stagegrowth procedure (i.e., 48 hrs MM, 96 hrs HGM). GC analyses showed thatall 12 strains produced more than 18% EPA of total lipids. Among them,strain #6 (designated as strain “Y2096”) produced about 24% EPA, strain#43 (designated as strain “Y2097”) produced about 22.3% EPA, strain #45(designated as strain “Y2098”) produced about 22.4% EPA, strain #47(designated as strain “Y2099”) produced about 22.6% EPA, strain #5produced about 23.3% EPA (designated as strain “Y2105”) and strain #48(designated as strain “Y2106”) produced about 23% EPA of total lipids.

The EPA content and the oil amount in strain Y2096 was further analyzedanalyzed using a modified two-stage growth procedure as follows. StrainY2096 was grown from a single colony in 3 mL SD+AA media at 30° C. withshaking at 250 rpm/min. After 24 hrs of growth, the 3 mL starter culturewas added to an Erlenmeyer flask containing 32 mL of SD+AA media. After48 hrs of additional growth at 30° C. and shaking at 250 rpm/min, thecells were pelleted and the supernatents were removed. The pellets werere-suspended in 35 mL HGM in a 250 mL flask. The 35 mL culture wasincubated at 30° C. and grown with shaking at 250 rpm/min for 4additional days. An aliquot (1 mL) of culture was used for GC analysisand 30 mL of culture was used for measurement of dry cell weight. GCanalysis was performed as described in the General Methods, except that40 μg of C15:0 (for use as an internal control) was added into sodiummethoxide for trans-esterification. Dry cell weight was determined bylyophilizing the H₂O-washed cell pellet from 30 mL culture.

GC analyses showed that Y2096 produced about 28.1% EPA of total lipids,with about 20.8% oil/dry cell weight. Strain Y2096 possessed thefollowing genotype with respect to wildtype Yarrowia lipolytica ATCC#20362: POX3-, LIP1-, Y.Δ12-, FBA::F.Δ12::Lip2, TEF::F.Δ12::Pex16,FBAIN::MΔ12::Pex20, TEF::Δ6S::Lip1, FBAIN::Δ6S::Lip1, FBAIN::E1S::Pex20,GPAT::E1S::Oct, GPDIN::E1S::Lip2, TEF::E2S::Xpr, FBAIN::MAΔ5::Pex20,TEF::MAΔ5::Lip1, TEF::HΔ5S::Pex16, TEF::IΔ5S::Pex20, GPAT::ID5S::Pex20,FBAIN::Δ17S::Lip2, FBAINm::Δ17S::Pex16, TEF::Δ17S::Pex16 and2×TEF::rELO2S::Pex20.

Generation of Y2098U Strain to Produce about 21% EPA of Total Lipidswith Ura- Phenotype

The SalI/PacI-digested construct pZKUT16 (SEQ ID NO:159) was used tointegrate a TEF::rELO2S::Pex20 chimeric gene into the Ura3 gene ofstrain Y2098 according to the General Methods. Following transformation,cells were plated onto MM+5-FOA selection plates and maintained at 30°C. for 2 to 3 days.

A total of 48 transformants grown on MM+5-FOA plates were picked andre-streaked onto MM plates and MM+5-FOA plates, separately.. The strainsthat could grow on MM+5-FOA plates, but not on MM plates, were selectedas Ura- strains. All of these 48 strains were analyzed using thetwo-stage growth conditions, as described in the General Methods (i.e.,48 hrs MMU, 96 hrs HGM). The cells were collected by centrifugation,lipids were extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed the presence of 12 to 20% EPA in all of thetransformants with pZKUT16 after the two-stage growth. One strain (i.e.,#33) produced about 21% EPA and was designated as strain “Y2098U”.

Generation of Y3000 Strain to Produce Greater than 5.6% DHA of TotalLipids

Construct pZP2FOEN4 (FIG. 9E, SEQ ID NO:165) was used to integrate acluster of two chimeric genes (comprising the synthetic C_(20/22)“OtE2S” elongase and synthetic Δ4 desaturase “D4S”) into the Pox2 genesite of strain Y2098U. Plasmid pZP2FOEN4 contained the followingcomponents:

TABLE 21 Description of Plasmid pZP2FOEN4 (SEQ ID NO: 165) RE Sites AndNucleotides Within Description Of Fragment And Chimeric Gene SEQ ID NO:165 Components BsiWI/AscI 810 bp 5′ part of Yarrowia Aco2 gene (GenBank(6152-6962) Accession No. AJ001300) SphI/EcoRI 655 bp 3′ part ofYarrowia Aco2 gene (GenBank (9670-10325) Accession No. AJ001300)EcoRI/PacI YAT::Δ4S::Pex16, comprising: (10640-2648) YAT promoter: YAT1promoter (SEQ ID NO: 217) Δ4S: codon-optimized Δ4 desaturase gene (SEQID NO: 106), derived from Thraustochytrium aureum (GenBank Accession No.AAN75707) Pex16: Pex16 terminator sequence of Yarrowia Pex16 gene(GenBank Accession No. U75433) EcoRI/SwaI with FBAIN::OtE2S::Oct,comprising: EcoRV FBAIN: FBAIN promoter (SEQ ID NO: 214) (10618-8345)OtE2S: codon-optimized OtE2S gene (SEQ ID NO: 102), derived fromOstreococcus tauri (GenBank Accession No. AY591336) OCT: OCT terminatorsequence of Yarrowia OCT gene (GenBank Accession No. X69988) SpeI/XbaIwith SpeI Yarrowia Ura3 gene (GenBank Accession (12582-11095) No.AJ306421)

The plasmid pZP2FOEN4 was digested with SphI/AscI, and then used totransform Y2098U strain according to the General Methods. Followingtransformation, the cells were plated onto MM selection media plates andmaintained at 30° C. for 2 to 3 days. A total of 24 transformants grownon the MM plates were picked and re-streaked onto fresh MM plates. Oncegrown, these strains were individually inoculated into 3 mL liquid MM at30° C. and shaken at 250 rpm/min for 2 days. The cells were collected bycentrifugation, lipids were extracted, and fatty acid methyl esters wereprepared by trans-esterification, and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed there were about 3.6 to 5.3% DPA and 0.4 to 1% DHA oftotal lipids produced in 12 of the 24 transformants. The 12 strainsproducing DPA and DHA were selected for further analyses using thetwo-stage growth procedure (i.e., 48 hrs MM, 96 hrs HGM). The cells werethen collected by centrifugation, lipids were extracted, and fatty acidmethyl esters were prepared by trans-esterification, and subsequentlyanalyzed with a Hewlett-Packard 6890 GC.

GC analyses showed there were 15.6 to 20% DPA and 2.9 to 5.6%. DHAproduced in these 12 transformants. The strain that produced 5.6% DHAwas designated as strain “Y3000”.

Example 7 Generation of Intermediate Strain Y2031, having A Ura-Genotype and Producing 45% LA of Total Lipids

Strain Y2031 was generated by integration of the TEF::Y.Δ12::Pex20chimeric gene of plasmid pKUNT2 (FIG. 10A) into the Ura3 gene locus ofwild type Yarrowia strain ATCC #20362, to thereby to generate aUra-genotype.

Specifically, plasmid pKUNT2 contained the following components:

TABLE 22 Description of Plasmid pKUNT2 (SEQ ID NO: 116) RE Sites AndNucleotides Within SEQ ID Description Of Fragment And Chimeric Gene NO:166 Components AscI/BsiWI 784 bp 5′ part of Yarrowia Ura3 gene (GenBank(3225-3015) Accession No. AJ306421) SphI/PacI 516 bp 3′ part of YarrowiaUra3 gene (GenBank (5933-13) Accession No. AJ306421) EcoRI/BsiWITEF::Y.Δ12::Pex20, comprising: (6380-8629) TEF: TEF promoter (GenBankAccession No. AF054508) Y.Δ12: Yarrowia Δ12 desaturase gene (SEQ ID NO:28) Pex20: Pex20 terminator sequence from Yarrowia Pex20 gene (GenBankAccession No. AF054613)

The pKUNT2 plasmid was digested with AscI/SphI, and then used fortransformation of wild type Y. lipolytica ATCC #20362 according to theGeneral Methods. The transformant cells were plated onto FOA selectionmedia plates and maintained at 30° C. for 2 to 3 days. The FOA resistantcolonies were picked and streaked onto MM and MMU selection plates. Thecolonies that could grow on MMU plates but not on MM plates wereselected as Ura- strains. Single colonies (5) of Ura- strains were theninoculated into liquid MMU at 30° C. and shaken at 250 rpm/min for 2days. The cells were collected by centrifugation, lipids were extracted,and fatty acid methyl esters were prepared by trans-esterification, andsubsequently analyzed with a Hewlett-Packard 6890 GC.

GC analyses showed that there were about 45% LA in two Ura- strains(i.e., strains #2 and #3), compared to about 20% LA in the wild typeATCC #20362. Transformant strain #2 was designated as strain “Y2031”.

Example 8 Synthesis and Functional Expression of A Codon-Optimized Δ9Elongase Gene in Yarrowia lipolytica

The codon usage of the Δ9 elongase gene of Isochrysis galbana (GenBankAccession No. AF3901 74) was optimized for expression in Y. lipolytica,in a manner similar to that described in WO 2004/101753 and Example 4.Specifically, according to the Yarrowia codon usage pattern, theconsensus sequence around the ATG translation initiation codon, and thegeneral rules of RNA stability (Guhaniyogi, G. and J. Brewer, Gene265(1-2):11-23 (2001)), a codon-optimized Δ9 elongase gene was designed(SEQ ID NO:71), based on the DNA sequence of the I. galbana gene (SEQ IDNO:69). In addition to modification of the translation initiation site,126 bp of the 792 bp coding region were modified (, and 123 codons wereoptimized. None of the modifications in the codon-optimized gene changedthe amino acid sequence of the encoded protein (GenBank Accession No.AF390174; SEQ ID NO:70).

In Vitro Synthesis of A Codon-Optimized Δ9 Elongase Gene for Yarrowia

The codon-optimized Δ9 elongase gene was synthesized as follows. First,eight pairs of oligonucleotides were designed to extend the entirelength of the codon-optimized coding region of the I. galbana Δ9elongase gene (e.g., IL3-1A, IL3-1B, IL3-2A, IL3-2B, IL3-3A, IL3-3B,IL3-4A, IL3-4B, IL3-5A, IL3-5B, IL3-6A, IL3-6B, IL3-7A, IL3-8A andIL3-8B, corresponding to SEQ ID NOs:292-307). Each pair of sense (A) andanti-sense (B) oligonucleotides were complementary, with the exceptionof a 4 bp overhang at each 5′-end. Additionally, primers IL3-1F, IL3-4R,IL3-5F and IL3-8R (SEQ ID NOs:308-311) also introduced NcoI, PstI, PstIand Not1 restriction sites, respectively, for subsequent subcloning.

Oligonucleotides (100 ng of each) were phosphorylated as described inExample 4, and then each pair of sense and antisense oligonucleotideswas mixed and annealed together [e.g., IL3-1A (SEQ ID NO:292) wasannealed to IL3-1B (SEQ ID NO:293) to produce the double-strandedproduct “IL3-1AB” and IL3-2A (SEQ ID NO:294) was annealed to IL3-2B (SEQID NO:295) to produce the double-stranded product “IL3-2AB”, etc.].

Two separate pools of annealed, double-stranded oligonucleotides werethen ligated together, as shown below: Pool 1 (comprising IL3-1AB,IL3-2AB, IL3-3AB and IL3-4AB); and, Pool 2 (comprising IL3-5AB, IL3-6AB,IL3-7AB and IL3-8AB). Each pool of annealed oligonucleotides was mixedin a volume of 20 μl with 10 U of T4 DNA ligase and the ligationreaction was incubated overnight at 16° C.

The product of each ligation reaction was then used as template toamplify the designed DNA fragment by PCR. Specifically, using theligated “Pool 1” mixture (i.e., IL3-1AB, IL3-2AB, IL3-3AB and IL3-4AB)as template, and oligonucleotides IL3-1F and IL3-4R (SEQ ID NOs:308 and309) as primers, the first portion of the codon-optimized Δ9 elongasegene was amplified by PCR. The PCR amplification was carried out in a 50μl total volume, as described in Example 4. The 417 bp PCR fragment wassubcloned into the pGEM-T easy vector (Promega) to generate pT9(1-4).

Using the ligated “Pool 2” mixture (i.e., IL3-5AB, IL3-6AB, IL3-7AB andIL3-8AB) as the template, and oligonucleotides IL3-5F and IL3-8R (SEQ IDNOs:310 and 311) as primers, the second portion of the codon-optimizedΔ9 elongase gene was amplified similarly by PCR and cloned intopGEM-T-easy vector to generate pT9(5-8).

E. coli was transformed separately with pT9(1-4) and pT9(5-8) and theplasmid DNA was isolated from ampicillin-resistant transformants.Plasmid DNA was purified and digested with the appropriate restrictionendonucleases to liberate the 417 bp NcoI/PstI fragment of pT9(1-4) (SEQID NO:312) and the 377 bp PstI/Not1 fragment of pT9(5-8) (SEQ IDNO:313). These two fragments were then combined and directionallyligated together with Nco1/Not1 digested pZUF17 (SEQ ID NO:162; FIG. 9B)to generate pDMW237 (FIG. 10B; SEQ ID NO:167). The DNA sequence of theresulting synthetic Δ9 elongase gene (“IgD9e”) in pDMW237 was exactlythe same as the originally designed codon-optimized gene (i.e., SEQ IDNO:71) for Yarrowia.

Expression of the Codon-Optimized Δ9 Elongase Gene in Y. lipolytica

Construct pDMW237 (FIG. 10B), an auto-replication plasmid comprising achimeric FBAIN::Ig D9e::Pex20 gene, was transformed into Y. lipolyticaY2031 strain (Example 7) as described in the General Methods. Threetransformants of Y2031 with pDMW237 were grown individually in MM mediafor two days and the cells were collected by centrifugation, lipids wereextracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

The GC results showed that there were about 7.1%, 7.3% and 7.4% EDA,respectively, produced in these transformants with pDMW237. These datademonstrated that the synthetic, codon-optimized IgD9e could convertC18:2 to EDA. The “percent (%) substrate conversion” of thecodon-optimized gene was determined to be about 13%.

Example 9 Synthesis of A Codon-Optimized Δ8 Desaturase Gene in Yarrowialipolytica

The codon usage of the Δ8 desaturase gene of Euglena gracilis (GenBankAccession No. AAD45877) was optimized for expression in Y. lipolytica,in a manner similar to that described in WO 2004/101753 and Examples 4and 8 (supra). Despite synthesis of three different codon-optimizedgenes (i.e., “D8S-1”, “D8S-2” and “D8S-3”), none of the genes werecapable of desaturating EDA to DGLA. It was therefore hypothesized thatthe previously published Δ8 desaturase sequences were incorrect and itwas necessary to isolate the Δ8 desaturase from Euglena gracilisdirectly, following mRNA isolation, cDNA synthesis and PCR. Thisresulted in two similar sequences, identified herein as Eg5 (SEQ IDNOs:77 and 78) and Eg12 (SEQ ID NOs:79 and 80).

Functional analysis of each gene sequence was performed by cloning thegenes into a Saccharomyces cerevisiae yeast expression vector andconducting substrate feeding trials. Although both Eg5 and Eg12 wereable to desaturase EDA and ETrA to produce DGLA and ETA, respectively,Eg5 had significantly greater activity than Eg12.

Based on the confirmed Δ8 desaturase activity of Eg5, the sequence wascodon-optimized for expression in Yarrowia lipolytica to thereby resultin the synthesis of a synthetic, functional codon-optimized Δ8desaturase designated as “D8SF” (SEQ ID NOs:81 and 82).

Preliminary In Vitro Synthesis of A Codon-Optimized Δ8 Desaturase Gene

A codon-optimized Δ8 desaturase gene (designated “D8S-1”; SEQ ID NO:75)was designed, based on the published sequence of Euglena gracilis (SEQID NOs:72 and 73), according to the Yarrowia codon usage pattern (WO2004/101753), the consensus sequence around the ‘ATG’ translationinitiation codon, and the general rules of RNA stability (Guhaniyogi, G.and J. Brewer, Gene 265(1-2):11-23 (2001)). In addition to modificationof the translation initiation site, 200 bp of the 1260 bp coding regionwere modified (15.9%). None of the modifications in the codon-optimizedgene changed the amino acid sequence of the encoded protein (SEQ IDNO:73) except the second amino acid from ‘K’ to ‘E’ to add a NcoI sitearound the translation initiation codon.

Specifically, the codon-optimized Δ8 desaturase gene was synthesized asfollows. First, thirteen pairs of oligonucleotides were designed toextend the entire length of the codon-optimized coding region of the E.gracilis Δ8 desaturase gene (e.g., D8-1A, D8-1B, D8-2A, D8-2B, D8-3A,D8-3B, D8-4A, D8-4B, D8-5A, D8-5B, D8-6A, D8-6B, D8-7A, D8-7B, D8-8A,D8-8B, D8-9A, D8-9B, D8-10A, D8-10B, D8-11B, D8-12A, D8-12B, D8-13A andD8-13B, corresponding to SEQ ID NOs:314-339). Each pair of sense (A) andanti-sense (B) oligonucleotides were complementary, with the exceptionof a 4 bp overhang at each 5′-end. Additionally, primers D8-1A, D8-3B,D8-7A, D8-9B and D8-13B (SEQ ID NOs:314, 319, 326, 331 and 339) alsointroduced NcoI, BgIII, Xho1, SacI and Not1 restriction sites,respectively, for subsequent subcloning.

Oligonucleotides (100 ng of each) were phosphorylated as described inExample 4, and then each pair of sense and antisense oligonucleotideswas mixed and annealed together [e.g., D8-1A (SEQ ID NO:314) wasannealed to D8-1B (SEQ ID NO:315) to produce the double- strandedproduct “D8-1AB” and D8-2A (SEQ ID NO:316) was annealed to D8-2B (SEQ IDNO:316) to produce the double-stranded product “D8-2AB”, etc.].

Four separate pools of annealed, double-stranded oligonucleotides werethen ligated together, as shown below: Pool 1 (comprising D8-1AB, D8-2ABand D8-3AB); Pool 2 (comprising D8-4AB, D8-5AB and D8-6AB); Pool 3(comprising D8-7AB, D8-8AB and D8-9AB); and, Pool 4 (comprising D8-10AB,D8-11AB, D8-12AB and D8-13AB). Each pool of annealed oligonucleotideswas mixed in a volume of 20 μl with 10 U of T4 DNA ligase and theligation reaction was incubated overnight at 16° C.

The product of each ligation reaction was then used as template toamplify the designed DNA fragment by PCR. Specifically, using theligated “Pool 1” mixture (i.e., D8-1AB, D8-2AB and D8-3AB) as template,and oligonucleotides D8-1F and D8-3R (SEQ ID NOs:340 and 341) asprimers, the first portion of the codon-optimized Δ8 desaturase gene wasamplified by PCR. The PCR amplification was carried out in a 50 μl totalvolume, as described in Example 4. The 309 bp PCR fragment was subclonedinto the pGEM-T easy vector (Promega) to generate pT8(1-3).

Using the ligated “Pool 2” mixture (i.e., D8-4AB, D8-5AB and D8-6AB) asthe template, and oligonucleotides D8-4F and D8-6R (SEQ ID NOs:342 and343) as primers, the second portion of the codon-optimized Δ8 desaturasegene was amplified similarly by PCR and cloned into pGEM-T-easy vectorto generate pT8(4-6). Using the ligated “Pool 3” mixture (i.e., D8-7AB,D8-8AB and D8-9AB) as the template and oligonucleotides D8-7F and D8-9R(SEQ ID NOs:344 and 345) as primers, the third portion of thecodon-optimized Δ8 desaturase gene was amplified similarly by PCR andcloned into pGEM-T-easy vector to generate pT8(7-9). Finally, using the“Pool 4” ligation mixture (i.e., D8-10AB, D8-11AB, D8-12AB and D8-13AB)as template, and oligonucleotides D8-10F and D8-13R (SEQ ID NOs:346 and347) as primers, the fourth portion of the codon-optimized Δ8 desaturasegene was amplified similarly by PCR and cloned into pGEM-T-easy vectorto generate pT8(10-13).

E. coli was transformed separately with pT8(1-3), pT8(4-6), pT8(7- 9)and pT8(10-13) and the plasmid DNA was isolated fromampicillin-resistant transformants. Plasmid DNA was purified anddigested with the appropriate restriction endonucleases to liberate the309 bp NcoI/BgIII fragment of pT8(1-3) (SEQ ID NO:348), the 321 bp//BgIII/XhoI fragment of pT8(4-6) (SEQ ID NO:349), the 264 bp XhoI/SacIfragment of pT8(7-9) (SEQ ID NO:350) and the 369 bp Sac1/Not1 fragmentof pT8(10-13) (SEQ ID NO:351). These fragments were then combined anddirectionally ligated together with Nco1/Not1 digested pY54PC (SEQ IDNO:168; WO2004/101757) to generate pDMW240 (FIG. 10C). This resulted ina synthetic Δ8 desaturase gene (“D8S-1”, SEQ ID NO:75) in pDMW240.

Compared with the published Δ8 desaturase amino acid sequence (SEQ IDNO:73) of E. gracilis, the second amino acid of D8S-1 was changed from‘K’ to ‘E’ in order to add the NcoI site around the translationinitiation codon. Another version of the synthesized gene, with theexact amino acid sequence as the published E. gracilis Δ8 desaturasesequence (SEQ ID NO:73), was constructed by in vitro mutagenesis(Stratagene, San Diego, Calif.) using pDMW240 as a template andoligonucleotides ODMW390 and ODMW391 (SEQ ID NOs:352 and 353) asprimers. The resulting plasmid was designated pDMW255. The synthetic Δ8desaturase gene in pDMW255 was designated as “D8S-2” and the amino acidsequence was exactly the same as the sequence depicted in SEQ ID NO:73.

Nonfunctional Codon-Optimized Δ8 Desaturase Genes

Yarrowia lipolytica strain ATCC #76982(Leu-) was transformed withpDMW240 (FIG. 10C) and pDMW255, respectively, as described in theGeneral Methods. Yeast containing the recombinant constructs were grownin MM supplemented with EDA [20:2(11,14)]. Specifically, single coloniesof transformant Y. lipolytica containing either pDMW240 (containingD8S-1) or pDMW255 (containing D8S-2) were grown in 3 mL MM at 30° C. toan OD₆₀₀˜1.0. For substrate feeding, 100 μl of cells were thensubcultured in 3 mL MM containing 10 μg of EDA substrate for about 24 hrat 30° C. The cells were collected by centrifugation, lipids wereextracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

Neither transformant produced DGLA from EDA and thus D8S-1 and D8S-2were not functional and could not desaturate EDA. The chimericD8S-1::XPR and D8S-2::XPR genes are shown in SEQ ID NOs:354 and 355,respectively.

A three amino acid difference between the protein sequence of the Δ8desaturase deposited in GenBank (Accession No. AAD45877 [SEQ ID NO:73])and in WO 00/34439 or Wallis et al. (Archives of Biochem. Biophys,365:307-316 (1999)) (SEQ ID NO:74 herein) was found. Specifically, threeamino acids appeared to be missing in GenBank Accession No. AAD45877.Using pDMW255 as template and ODMW392 and ODMW393 (SEQ ID NOs:356 and357) as primers, 9 bp were added into the synthetic D8S-2 gene by invitro mutagenesis (Stratagene, San Diego, Calif.), thus producing aprotein that was identical to the sequence described in WO 00/34439 andWallis et al. (supra) (SEQ ID NO:74). The resulting plasmid was calledpDMW261. The synthetic Δ8 desaturase gene in pDMW261 was designated as“D8S-3” (SEQ ID NO:76). Following transformation of the pDMW261construct into Yarrowia, a similar feeding experiment using EDA wasconducted, as described above. No desaturation of EDA to DGLA wasobserved with D8S-3.

Isolation of A Euglena gracilis Δ8 Desaturase Gene

Euglena gracilis was obtained from Dr. Richard Triemer's lab at MichiganState University (East Lansing, Mich.). From 10 mL of actively growingculture, a 1 mL aliquot was transferred into 250 mL of Euglena gracilis(Eg) Medium in a 500 mL glass bottle. Eg medium was made by combining: 1g of sodium acetate, 1 g of beef extract (Catalog #U126-01, DifcoLaboratories, Detroit, Mich.), 2 g of Bacto®Tryptone (Catalog#0123-17-3, Difco Laboratories) and 2 g of Bacto®Yeast Extract (Catalog#0127-17-9, Difco Laboratories) in 970 mL of water. After filtersterilizing, 30 mL of Soil-Water Supernatant (Catalog #15-3790, CarolinaBiological Supply Company, Burlington, N.C.) was aseptically added toproduce the final Eg medium. E. gracilis cultures were grown at 23° C.with a 16 hr light, 8 hr dark cycle for 2 weeks with no agitation.

After 2 weeks, 10 mL of culture was removed for lipid analysis andcentrifuged at 1,800×g for 5 min. The pellet was washed once with waterand re-centrifuged. The resulting pellet was dried for 5 min undervacuum, resuspended in 100 μL of trimethylsulfonium hydroxide (TMSH) andincubated at room temperature for 15 min with shaking. After this, 0.5mL of hexane was added and the vials were incubated for 15 min at roomtemperature with shaking.. Fatty acid methyl esters (5 μL injected fromhexane layer) were separated and quantified using a Hewlett-Packard 6890Gas Chromatograph fitted with an Omegawax 320 fused silica capillarycolumn (Catalog #24152, Supelco Inc.). The oven temperature wasprogrammed to hold at 220° C. for 2.7 min, increase to 240° C. at 20°C./min and then hold for an additional 2.3 min. Carrier gas was suppliedby a Whatman hydrogen generator. Retention times were compared to thosefor methyl esters of standards commercially available (Catalog #U-99-A,Nu-Chek Prep, Inc.) and the resulting chromatogram is shown in FIG. 11.

The remaining 2 week culture (240 mL) was pelleted by centrifugation at1,800×g for 10 min, washed once with water and re-centrifuged. Total RNAwas extracted from the resulting pellet using the RNA STAT-60™ reagent(TEL-TEST, Inc., Friendswood, Tex.) and following the manufacturer'sprotocol provided (use 5 mL of reagent, dissolved RNA in 0.5 mL ofwater). In this way, 1 mg of total RNA (2 mg/mL) was obtained from thepellet. The mRNA was isolated from 1 mg of total RNA using the mRNAPurification Kit (Amersham Biosciences, Piscataway, N.J.) following themanufacturer's protocol provided. In this way, 85 μg of mRNA wasobtained.

cDNA was synthesized from 765 ng of mRNA using the SuperScript™ ChoiceSystem for cDNA synthesis (Invitrogen™ Life Technologies, Carlsbad,Calif.) with the provided oligo(dT) primer according to themanufacturer's protocol. The synthesized cDNA was dissolved in 20 μL ofwater.

The E. gracilis Δ8 desaturase was amplified from cDNA witholigonucleotide primers Eg5-1 and Eg3-3 (SEQ ID NOs:358 and 359) usingthe conditions described below. Specifically, cDNA (1 μL) was combinedwith 50 pmol of Eg5-1, 50 pmol of Eg5-1, 1 μL of PCR nucleotide mix (10mM, Promega, Madison, Wis.), 5 μL of 10×PCR buffer (Invitrogen), 1.5 μLof MgCl₂ (50 mM, Invitrogen), 0.5 μL of Taq polymerase (Invitrogen) andwater to 50 μL. The reaction conditions were 94° C. for 3 min followedby 35 cycles of 94° C. for 45 sec, 55° C. for 45 sec and 72° C. for 1min. The PCR was finished at 72° C. for 7 min and then held at 4° C. ThePCR reaction was analyzed by agarose gel electrophoresis on 5 μL and aDNA band with molecular weight around 1.3 kB was observed. The remaining45 μL of product was separated by agarose gel electrophoresis and theDNA band was purified using the Zymoclean™ Gel DNA Recovery Kit (ZymoResearch, Orange, Calif.) following the manufacturer's protocol. Theresulting DNA was cloned into the pGEM®-T Easy Vector (Promega)following the manufacturer's protocol. Multiple clones were sequencedusing T7, M13-28Rev, Eg3-2 and Eg5-2 (SEQ ID NOS:360-363, respectively).

Thus, two classes of DNA sequences were obtained, Eg5 (SEQ ID NO:77) andEg12 (SEQ ID NO:79), that differed in only a few bp. Translation of Eg5and Eg12 gave rise to protein sequences that differed in only one aminoacid, SEQ ID NO:78 and 80, respectively. Thus, the DNA and proteinsequences for Eg5 are set forth in SEQ ID NO:77 and SEQ ID NO:78,respectively; the DNA and protein sequences for Eg12 are set forth inSEQ ID NO:79 and SEQ ID NO:80, respectively.

Comparison of the Isolated E. gracilis Δ8 Desaturase Sequences toPublished E. gracilis Δ8 Desaturase Sequences

An alignment of the protein sequences set forth in SEQ ID NO:78 (Eg5)and SEQ ID NO:80 (Eg12) with the protein sequence from GenBank AccessionNo. AAD45877 (gi: 5639724; SEQ ID NO:73 herein) and with the publishedprotein sequences of Wallis et al. (Archives of Biochem. Biophys.,365:307-316 (1999); WO 00/34439) [SEQ ID NO:74 herein] is shown in FIG.12. Amino acids conserved among all 4 sequences are indicated with anasterisk (*). Dashes are used by the program to maximize alignment ofthe sequences. The putative cytochrome b₅ domain is underlined. Aputative His box is shown in bold. Percent identity calculationsrevealed that the Eg5 Δ8 desaturase protein sequence is 95.5% identicalto SEQ ID NO:73 and 96.2% identical to SEQ ID NO:74, wherein “%identity” is defined as the percentage of amino acids that are identicalbetween the two proteins. Sequence alignments and percent identitycalculations were performed using the Megalign program of the LASERGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiplealignment of the sequences was performed using the Clustal method ofalignment (Higgins and Sharp, CABIOS. 5:151-153 (1989)) with defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parametersfor pairwise alignments using the Clustal method were KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For a more complete analysisof the differences between the various E. gracilis Δ8 desaturasesequences, refer to co-pending U.S. patent application Ser. No.11/166993.

Functional Analysis of the Euglena gracilis Δ8 Desaturase Sequences inSaccharomyces cerevisiae

The yeast episomal plasmid (YEp)-type vector pRS425 (Christianson etal., Gene, 110:119-22 (1992)) contains sequences from the Saccharomycescerevisiae 2μ endogenous plasmid, a LEU2 selectable marker and sequencesbased on the backbone of a multifunctional phagemid, pBluescript II SK+.The S. cerevisiae strong, constitutive glyceraldehyde-3-phosphatedehydrogenase (GPD) promoter was cloned between the SacII and SpeI sitesof pRS425 in the same way as described in Jia et al. (PhysiologicalGenomics, 3:83-92 (2000)) to produce pGPD-425. A NotI site wasintroduced into the BamHI site of pGPD-425 (thus producing a NotI siteflanked by BamHI sites), thereby resulting in plasmid pY-75. Eg5 (SEQ IDNO:77) and Eg12 (SEQ ID NO:79) were released from the pGEM®-T Easyvectors described above by digestion with NotI and cloned into the NotIsite of pY-75 to produce pY89-5 (deposited as ATCC #PTA-6048) andpY89-12, respectively. In this way, the Δ8 desaturases (i.e., Eg5 [SEQID NO:77] and Eg12 [SEQ ID NO:79]) were cloned behind a strongconstitutive promoter for expression in S. cerevisiae. A map of pY89-5is shown in FIG. 10D.

Plasmids pY89-5, pY89-12 and pY-75 were transformed into Saccharomycescerevisiae BY4741 (ATCC #201388) using standard lithium acetatetransformation procedures. Transformants were selected on DOBA mediasupplemented with CSM-leu (Qbiogene, Carlsbad, Calif.). Transformantsfrom each plate were inoculated into 2 mL of DOB medium supplementedwith CSM-leu (Qbiogene) and grown for 1 day at 30° C., after which 0.5mL was transferred to the same medium supplemented with either EDA orEtrA to 1 mM. These were incubated overnight at 30° C., 250 rpm, andpellets were obtained by centrifugation and dried under vacuum. Pelletswere transesterified with 50 μL of TMSH and analyzed by GC as describedin the General Methods. Two clones for pY- 75 (i.e., clones 75-1 and75-2) and pY89-5 (i.e., clones 5-6-1 and 5-6-2) were each analyzed,while two sets of clones for pY89-12 (i.e., clones 12-8-1, 12-8-2,12-9-1 and 12-9-2) from two independent transformations were analyzed.

The lipid profile obtained by GC analysis of clones fed EDA are shown inTable 23; and the lipid profile obtained by GC analysis of clones fedEtrA are shown in Table 24. Fatty acids are identified as 16:0(palmitate), 16:1 (palmitoleic acid), 18:0, 18:1 (oleic acid), 20:2[EDA], 20:3 (8,11,14) [DGLA], 20:3 (11,14,17) [ETrA] and 20:4(8,11,14,17) [ETA]; and the composition of each is presented as a % ofthe total fatty acids.

TABLE 23 Lipid Analysis Of Transformant S. cerevisiae Overexpressing TheEuglena gracilis Δ8 Desaturases: EDA Substrate Feeding 20:3 (8, 11, %20:2 Clone 16:0 16:1 18:0 18:1 20:2 14) Converted 75-1 (control) 14 32 538 10 0 0 75-2 (control) 14 31 5 41 9 0 0 5-6-1 (Eg5) 14 32 6 40 6 2 245-6-2 (Eg5) 14 30 6 41 7 2 19 12-8-1 (Eg12) 14 30 6 41 9 1 7 12-8-2(Eg12) 14 32 5 41 8 1 8 12-9-1 (Eg12) 14 31 5 40 9 1 8 12-9-2 (Eg12) 1432 5 41 8 1 7

TABLE 24 Lipid Analysis Of Transformant S. cerevisiae Overexpressing TheEuglena gracilis Δ8 Desaturases: ETrA Substrate Feeding 20:3 (11, 20:414, (8, 11, % 20:3 Clone 16:0 16:1 18:0 18:1 17) 14, 17) Converted 75-1(control) 12 25 5 33 24 0 0 75-2 (control) 12 24 5 36 22 1 5 5-6-1 (Eg5)13 25 6 34 15 7 32 5-6-2 (Eg5) 13 24 6 34 17 6 27 12-8-1 (Eg12) 12 24 534 22 2 8 12-8-2 (Eg12) 12 25 5 35 20 2 9 12-9-1 (Eg12) 12 24 5 34 22 29 12-9-2 (Eg12) 12 25 6 35 20 2 9

The data in Tables 23 and 24 showed that the cloned Euglena Δ8desaturases were able to desaturate EDA and EtrA. The sequence set forthin SEQ ID NO:80 has one amino acid change compared to the sequence setforth in SEQ ID NO:78 and has reduced Δ8 desaturase activity.

The small amount of 20:4(8,11,14,17) generated by clone 75-2 in Table 24had a slightly different retention time than a standard for20:4(8,11,14,17). This peak was more likely a small amount of adifferent fatty acid generated by the wild-type yeast in thatexperiment.

Further Modification of the Δ8 Desaturase Gene Codon-Optimized forYarrowia lipolytica

The amino acid sequence of the synthetic D8S-3 gene in pDMW261 wascorrected according to the amino acid sequence of the functional EuglenaΔ8 desaturase (SEQ ID NOs:77 and 78). Using pDMW261 as a template andoligonucleotides ODMW404 (SEQ ID NO:364) and D8-13R (SEQ ID NO:347), theDNA fragment encoding the synthetic D8S-3 desaturase gene was amplified.The resulting PCR fragment was purified with Bio101's Geneclean kit andsubsequently digested with Kpn1 and Not1 (primer ODMW404 introduced aKpnI site while primer D8-13R introduced a NotI site). The Kpn1/Not1fragment (SEQ ID NO:365) was cloned into Kpn1/Not1 digested pKUNFmKF2(FIG. 10E; SEQ ID NO:169) to produce pDMW277 (FIG. 13A).

Oligonucleotides YL521 and YL522 (SEQ ID NOs:366 and 367), which weredesigned to amplify and correct the 5′ end of the D8S-3 gene, were usedas primers in another PCR reaction where pDMW277 was used as thetemplate. The primers introduced into the PCR fragment a Nco1 site andBgIII site at its 5′ and 3′ ends, respectively. The 318 bp PCR productwas purified with Bio101's GeneClean kit and subsequently digested withNco1 and BgIII. The digested fragment, along with the 954 bp BgIII/NotIfragment from pDMW277, was used to exchange the NcoI/NotI fragment ofpZF5T-PPC (FIG. 13B; SEQ ID NO:170) to form pDMW287. In addition tocorrecting the 5′ end of the synthetic D8S-3 gene, this cloning reactionalso placed the synthetic Δ8 desaturase gene under control of theYarrowia lipolytica FBAIN promoter (SEQ ID NO:214).

The first reaction in a final series of site-directed mutagenesisreactions was then performed on pDMW287. The first set of primers, YL525and YL526 (SEQ ID NOs:368 and 369), was designed to correct amino acidfrom F to S (position #50) of the synthetic D8S-3 gene in pDMW287. Theplasmid resulting from this mutagenesis reaction then became thetemplate for the next site-directed mutagenesis reaction with primersYL527 and YL528 (SEQ ID NOs:370 and 371). These primers were designed tocorrect the amino acid from F to S (position #67) of the D8S-3 gene andresulted in creation of plasmid pDMW287/YL527.

To complete the sequence corrections within the second quarter of thegene, the following reactions were carried out concurrently with themutations on the first quarter of the gene. Using pDMW287 as templateand oligonucleotides YL529 and YL530 (SEQ ID NOs:372 and 373) asprimers, an in vitro mutagenesis reaction was carried out to correct theamino acid from C to W (position #177) of the synthetic D8S-3 gene. Theproduct (i.e., pDMW287/Y529) of this mutagenesis reaction was used asthe template in the following reaction using primers YL531 and YL532(SEQ ID NOs:374 and 375) to correct the amino acid from P to L (position#213). The product of this reaction was called pDMW287/YL529-31.

Concurrently with the mutations on the first and second quarter of thegene, reactions were similarly carried out on the 3′ end of the gene.Each subsequent mutagenesis reaction used the plasmid product from thepreceding reaction. Primers YL533 and YL534 (SEQ ID NOs:376 and 377)were used on pDMW287 to correct the amino acid from C to S (position#244) to create pDMW287/YL533. Primers YL535 and YL536 (SEQ ID NOs:378and 379) were used to correct the amino acid A to T (position #280) inthe synthetic D8S-3 gene of pDMW287/YL533 to form pDMW287/YL533-5.Finally, the amino acid P at position #333 was corrected to S in thesynthetic D8S-3 gene using pDMW287/YL533-5 as the template and YL537 andYL538 (SEQ ID NOs:380 and 381) as primers. The resulting plasmid wasnamed pDMW287/YL533-5-7.

The BgIII/XhoI fragment of pDMW287/YL529-31 and the XhoI/NotI fragmentof pDMW287/YL533-5-7 were used to change the BgIII/NotI fragment ofpDMW287/YL257 to produce pDMW287F (FIG. 13C) containing the completelycorrected synthetic Δ8 desaturase gene, designated D8SF and set forth inSEQ ID NO:81. SEQ ID NO:82 sets forth the amino acid sequence encoded bynucleotides 2-1270 of SEQ ID NO:81, which is essentially the same as thesequence set forth in SEQ ID NO:78, except for an additional valinefollowing the start methionine.

Example 10 Functional Expression of the Codon-Optimized Δ9 Elongase Geneand Codon-Optimized Δ8 Desaturase in Yarrowia lipolytica

The present Example describes DGLA biosynthesis and accumulation inYarrowia lipolytica that was transformed to co-express thecodon-optimized Δ9 elongase and codon-optimized Δ8 desaturase fromExamples 8 and 9. This experiment thereby confirmed both genes' activityand Y. lipolytica's ability to express the Δ9 elongase/Δ8 desaturasepathway.

Specifically, the ClaI/PacI fragment comprising a chimericFBAIN::D8SF::Pex16 gene of construct pDMW287F (Example 9) was insertedinto the ClaI/PacI sites of pDMW237 (Example 8) to generate theconstruct pDMW297 (FIG. 13D; SED ID NO:123).

Plasmid pDMW297 contained the following components:

TABLE 25 Description of Plasmid pDMW297 (SEQ ID NO: 171) RE Sites AndNucleotides Within SEQ ID Description Of Fragment And Chimeric Gene NO:171 Components EcoRI/ClaI ARS18 sequence (GenBank Accession No. A17608)(9053-10448) ClaI/PacI FBAIN::D8SF::Pex16, comprising: (1-2590) FBAIN:FBAIN promoter (SEQ ID NO: 214) D8SF: codon-optimized Δ8 desaturase gene(SEQ ID NO: 81), derived from Euglena gracilis (GenBank Accession No.AF139720) Pex16: Pex16 terminator sequence of Yarrowia Pex16 gene(GenBank Accession No. U75433) PacI/SalI Yarrowia Ura3 gene (GenBankAccession No. (2590-4082) AJ306421) SalI/BsiWI FBAIN::IgD9e::Pex20,comprising: (4082-6257) FBAIN: FBAIN promoter (SEQ ID NO: 214) IgD9e:codon-optimized Δ9 elongase gene (SEQ ID NO: 71), derived fromIsochrysis galbana (GenBank Accession No. 390174) Pex20: Pex20terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613)

Construct pDMW297 was then used for transformation of strain Y2031(Example 7) according to the General Methods. The transformant cellswere plated onto MM selection media plates and maintained at 30° C. for2 to 3 days. A total of 8 transformants grown on the MM plates werepicked and re-streaked onto fresh MM plates. Once grown, these strainswere individually inoculated into liquid MM at 30° C. and shaken at 250rpm/min for 2 days. The cells were collected by centrifugation, lipidswere extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed that DGLA was produced in all of the transformantsanalyzed. One strain produced about 3.2%, 4 strains produced 4.3-4.5%,two strains produced 5.5-5.8% and one strain produced 6.4% DGLA(designated herein as strain “Y0489”). The “percent (%) substrateconversion” of the codon-optimized D8SF gene in strain Y0489 wasdetermined to be 75%.

Example 11 The ω-6 Δ9 Elongase/Δ8 Desaturase Pathway: Generation ofStrains to Produce DHA In Yarrowia lipolytica (Prophetic)

The present Example describes the construction of strains derived fromYarrowia lipolytica ATCC #20362, engineered to produce DHA viaexpression of the ω-6 Δ9 elongase/Δ8 desaturase pathway; thus, analysisof the complete lipid profiles of these DHA-producing strains wouldindicate no GLA co-synthesis in the final DHA-containing oil.

The development of these DHA-producing strains first required creationof strains Y2201 and Y2203, derived from Yarrowia lipolytica ATCC#20362, capable of producing 9% EPA relative to the total lipids (FIG.5). Strains Y2201 and Y2203 required the construction of strains Y2152and Y2153 (producing ˜3.5% DGLA), strain Y2173 (producing 14% DGLA) andstrain Y2189 (producing 5% EPA).

Subsequently, since Y2201 and Y2203 are Lys-strains, it would benecessary to exchange the Yarrowia Ura3 gene (GenBank Accession No.AJ306421) spanning nucleotide positions 12582-11095 of pZP2FOEN4 (SEQ IDNO:165) with the Yarrowia Lys5 gene (GenBank Accession No. M34929) tocreate plasmid pZP2FOEN4-Lys, using methodology well known to one ofskill in the art. Following this manipulation, strains Y2201 and Y2203could be transformed with plasmid pZP2FOEN4-Lys, as described above inExample 6, thereby resulting in a transformant strain producing DHAusing the ω-6 Δ9 elongase/Δ8 desaturase pathway.

Generation of Strains Y2152 and Y2153 to Produce about ˜3.5% DGLA ofTotal Lipids

Construct pZP2C16M899 (FIG. 14A, SEQ ID NO:172) was used to integrate acluster of four chimeric genes (comprising two Δ9 elongases, a syntheticC_(16/18) fatty acid elongase and a Δ8 desaturase), as well as aYarrowia AHAS gene (acetohydroxy-acid synthase) containing a singleamino acid mutation. The mutated AHAS enzyme in Yarrowia conferredresistance to sulfonylurea, which was used as a positive screeningmarker. Plasmid pZP2C16M899 was designed to integrate into the Pox2 genesite of Yarrowia strain ATCC #20362 and thus contained the followingcomponents:

TABLE 26 Description of Plasmid pZP2C16M899 (SEQ ID NO: 172) RE SitesAnd Nucleotides Within SEQ ID Description Of Fragment And Chimeric GeneNO: 172 Components BsiWI/AscI 810 bp 5′ part of Yarrowia Aco2 gene(GenBank (6152-6962) Accession No. AJ001300) SphI/EcoRI 655 bp 3′ partof Yarrowia Aco2 gene (GenBank (9670-10325) Accession No. AJ001300)BsiWI/PmeI with GPM/FBAintron::rELO2S::Oct, comprising: EcoRV GPM/FBAIN:GPM::FBAIN chimeric promoter (SEQ (929-3195) ID NO: 219) rELO2S:codon-optimized rELO2 elongase gene (SEQ ID NO: 85), derived from rat(GenBank Accession No. AB071986) OCT: OCT terminator sequence ofYarrowia OCT gene (GenBank Accession No. X69988) BsiWI/EcoRIGPAT::IgD9e::Pex20, comprising: (929-14447, GPAT: GPAT promoter (SEQ IDNO: 216) reverse) IgD9e: codon-optimized Δ9 elongase gene (SEQ ID NO:71), derived from I. galbana Pex20: Pex20 terminator sequence ofYarrowia Pex20 gene (GenBank Accession No. AF054613) EcoRI/SwaITEF::IgD9e::Lip1, comprising: (14447-12912) TEF: TEF promoter (GenBankAccession No. AF054508) IgD9e: SEQ ID NO: 71 (supra) Lip1: Lip1terminator sequence of Yarrowia Lip1 gene (GenBank Accession No. Z50020)SwaI/PacI FBAIN::D8SF::Pex16, comprising: (12912-10325) FBAIN: FBAINpromoter (SEQ ID NO: 214) D8SF: codon-optimized Δ8 desaturase gene (SEQID NO: 81), derived from Euglena gracilis (GenBank Accession No.AF139720) Pex16: Pex16 terminator sequence of Yarrowia Pex16 gene(GenBank Accession No. U75433) gene PmeI with Yarrowia lipolytica AHASgene comprising a W497L EcoRV/ mutation (SEQ ID NO: 243) BsiWI(3195-6152)

Plasmid pZP2C16M899 was digested with SphI/AscI, and then used totransform ATCC #20362 according to the General Methods. Followingtransformation, cells were plated onto MM plates containing 150 mgsulfonylurea and maintained at 30° C. for 2 to 3 days. The sulfonylurearesistant colonies were picked and streaked onto MM with sulfonylureaselection plates. A total of 96 transformants were then inoculated intoliquid MM with sulfonylurea at 30° C. and shaken at 250 rpm/min for 2days. The cells were collected by centrifugation, lipids were extracted,and fatty acid methyl esters were prepared by trans-esterification, andsubsequently analyzed with a Hewlett-Packard 6890 GC.

GC analyses showed the presence of DGLA in the transformants containingthe 4 chimeric genes of pZP2C16M899, but not in the wild type Yarrowiacontrol strain. Most of the selected 96 strains produced less than 2%DGLA of total lipids. There were 28 strains that produced 2-2.9% DGLA oftotal lipids. There were 2 strains that produced about 3.5% DGLA oftotal lipids. Strains #65 and #73 were designated herein as strains“Y2152” and “Y2153”, respectively.

Generation of Strains Y2173 and Y2175 to Produce about 14-16% DGLA ofTotal Lipids

Construct pDMW314 (FIG. 14B, SEQ ID NO:173) was used to integrate acluster of four chimeric genes (comprising two Δ9 elongases, a Δ8desaturase and a Δ12 desaturase) into the Ura3 gene site of Yarrowiastrains Y2152 and Y2153, to thereby enhance production of DGLA. PlasmidpDMW314 contained the following components:

TABLE 27 Description of Plasmid pDMW314 (SEQ ID NO: 173) RE Sites AndNucleotides Within SEQ ID Description Of Fragment And Chimeric Gene NO:173 Components AscI/BsiWI 784 bp 5′ part of Yarrowia Ura3 gene (GenBank(10066-9275) Accession No. AJ306421) SphI/PacI 516 bp 3′ part ofYarrowia Ura3 gene (GenBank (12774-1) Accession No. AJ306421) SwaI/BsiWIFBAIN::F.D12S::Pex20, comprising: (6582-9275) FBAIN: FBAIN promoter (SEQID NO: 214) F.Δ12: Fusarium moniliforme Δ12 desaturase gene (SEQ ID NO:32) Pex20: Pex20 terminator sequence from Yarrowia Pex20 gene (GenBankAccession No. AF054613) ClaI/EcoRI GPAT::IgD9E::Pex20: as described forpZP2C16M899 (6199-4123) (supra) EcoRI/SwaI TEF:: IgD9E::Lip1: asdescribed for pZP2C16M899 (4123-2588) (supra) SwaI/PacIFBAIN::D8SF::Pex16: as described for pZP2C16M899 (2588-1) (supra)

Plasmid pDMW314 was digested with AscI/SphI, and then used fortransformation of Y. lipolytica strains Y2152 and Y2153 according to theGeneral Methods. The transformant cells were plated onto FOA selectionmedia plates and maintained at 30° C. for 2 to 3 days. The FOA resistantcolonies were picked and streaked onto MM and MMU selection plates. Thecolonies that could grow on MMU plates but not on MM plates wereselected as Ura- strains. Single colonies of Ura- strains were theninoculated into liquid MMU at 30° C. and shaken at 250 rpm/min for 2days. The cells were collected by centrifugation, lipids were extracted,and fatty acid methyl esters were prepared by trans-esterification, andsubsequently analyzed with a Hewlett-Packard 6890 GC.

GC analyses showed increased production of DGLA in almost alltransformants containing the 4 chimeric genes of pDMW314. Most of theselected 48 Ura⁻ strains of Y2152 with pDMW314 produced about 6-8% DGLAof total lipids. There was one strain (i.e., #47, designated herein as“Y2173”) that produced about 13.9% DGLA of total lipids.

Similarly, most of the selected 24 Ura⁻ strains of Y2153 with pDMW314produced about 6-8% DGLA of total lipids. There were two strains (i.e.,#6 and #11, designated herein as strains “Y2175” and “Y2176”) thatproduced about 16.3% and 17.2% DGLA of total lipids, respectively.

Generation of Strain Y2189 to Produce about 4.8% EPA of Total Lipids

Construct pDMW325 (FIG. 14C, SEQ ID NO:174) was used to integrate acluster of four chimeric genes (comprising two Δ5 desaturases and twoΔ17 desaturases) into the Leu2 gene site of Yarrowia Y2173 strain tothereby enable production of EPA. Plasmid pDMW325 contained thefollowing components:

TABLE 28 Description Of Plasmid pDMW325 (SEQ ID NO: 174) RE Sites AndNucleotides Within SEQ ID Description Of Fragment And Chimeric Gene NO:174 Components AscI/BsiWI 788 bp 5′ part of Yarrowia Leu2 gene (GenBank(4837-5632) Accession No. AF260230) SphI/PacI 703 bp 3′ part of YarrowiaLeu2 gene (GenBank (2137-1426) Accession No. AF260230) SwaI withFBAIN::MAΔ5::Pex20, comprising: Pme/BsiWI FBAIN: FBAIN promoter (SEQ IDNO: 214) (8277-5632) MAΔ5: Mortierella alpina Δ5 desaturase gene (SEQ IDNO: 6) (GenBank Accession No. AF067654) Pex20: Pex20 terminator sequenceof Yarrowia Pex20 gene (GenBank Accession No. AF054613) EcoRI/SwaIGPM/FBAIN::I.Δ5S::Oct, comprising: with PmeI GPM/FBAIN: GPM::FBAINchimeric promoter (SEQ ID (10876-8278) NO: 219) I.Δ5S: codon-optimizedΔ5 desaturase gene (SEQ ID NO: 10), derived from Isochrysis galbana (WO2002/ 081668) OCT: OCT terminator sequence of Yarrowia OCT gene (GenBankAccession No. X69988) EcoRI/PmeI Yarrowia Ura3 gene (GenBank AccessionNo. (10876-12497) AJ306421) PmeI/ClaI YAT::D17S::Lip2, comprising:(12497-14651 YAT: YAT1 promoter (SEQ ID NO: 217) Δ17S: codon-optimizedΔ17 desaturase gene (SEQ ID NO: 21), derived from S. diclina Lip2: Lip2terminator of Yarrowia lipase2 gene (GenBank Accession No. AJ012632)ClaI/PacI GPD::D17S::Pex16, comprising: (14651-1426 GPD: GPD promoter(SEQ ID NO: 210) Δ17S: SEQ ID NO: 21 (supra) Pex16: Pex16 terminatorsequence of Yarrowia Pex16 gene (GenBank Accession No. U75433)

Plasmid pDMW325 was digested with AscI/SphI, and then used to transformstrain Y2173 according to the General Methods. Following transformation,the cells were plated onto MMLe plates and maintained at 30° C. for 2 to3 days. The individual colonies grown on MMLe plates from eachtransformation were picked and streaked onto MM and MMLe plates. Thosecolonies that could grow on MMLe plates but not on MM plates wereselected as Leu2⁻ strains. Single colonies of Leu2⁻ strains were theninoculated into liquid MMLe media at 30° C. and shaken at 250 rpm/minfor 2 days. The cells were collected by centrifugation, lipids wereextracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed the presence of EPA in pDMW325 transformants, but notin the parental Y2173 strain. Specifically, among the 48 selected Leu2⁻transformants of Y2173 with pDMW325, most strains produced less than 3%EPA of total lipids. There were two strains (i.e., #21 and #46,designated herein as “Y2189” and “Y2190”) that produced about 4.8% and3.4% EPA of total lipids, respectively.

Generation of Strains Y2201 and Y2203 to Produce about 9% EPA of TotalLipids

Construct pZKSL5598 (FIG. 14D, SEQ ID NO:175) was used to integrate acluster of four chimeric genes (comprising a Δ9 elongase, a Δ8desaturase and two Δ5 desaturases) into the Lys5 gene (GenBank AccessionNo. M34929) site of Yarrowia Y2189 strain to thereby enhance productionof EPA. Plasmid pZKSL5598 contained the following components:

TABLE 29 Description of Plasmid pZKSL5598 (SEQ ID NO: 175) RE Sites AndNucleotides Within SEQ ID Description Of Fragment And Chimeric Gene NO:175 Components AscI/BsiWI 794 bp 5′ part of Yarrowia Lys5 gene (GenBank(10409-9573) Accession No. M34929) SphI/PacI 687 bp 3′ part of YarrowiaLys5 gene (GenBank (13804-13117) Accession No. M34929) BsiWI/SwaINT::I.D5S::Lip1, comprising: (7150-9573) NT: YAT1 promoter (SEQ ID NO:217) I.Δ5S: codon-optimized Δ5 desaturase gene (SEQ ID NO: 10), derivedfrom Isochrysis galbana (WO 2002/ 081668) Lip1: Lip1 terminator sequenceof Yarrowia Lip1 gene (GenBank Accession No. Z50020) SalI/BsiWIGPAT::MAΔ5::Pex20, comprising: (4537-7150) GPAT: GPAT promoter (SEQ IDNO: 216) MAΔ5: Mortierella alpina Δ5 desaturase gene (SEQ ID NO: 6)(GenBank Accession No. AF067654) Pex20: Pex20 terminator sequence fromYarrowia Pex20 gene (GenBank Accession No. AF054613) SwaI/PmeIFBAINm::IgD9e::OCT, comprising: (2381-348) FBAINm: FBAINm promoter (SEQID NO: 215) IgD9e: codon-optimized Δ9 elongase gene (SEQ ID NO: 71),derived from I. galbana OCT: OCT terminator sequence of Yarrowia OCTgene (GenBank Accession No. X69988) ClaI/PacI GPD::D8SF::Pex16,comprising: (1-13804) GPD: GPD promoter (SEQ ID NO: 210) D8SF:codon-optimized Δ8 desaturase gene (SEQ ID NO: 81), derived from Euglenagracilis (GenBank Accession No. AF139720) Pex16: Pex16 terminatorsequence of Yarrowia Pex16 gene (GenBank Accession No. U75433)SalII/PmeI Yarrowia Leu2 gene (GenBank Accession No. (4537-2417)AF260230)

Plasmid pZKSL5598 was digested with AscI/SphI, and then used totransform strain Y2189 according to the General Methods. Followingtransformation, the cells were plated onto MMLys plates and maintainedat 30° C. for 2 to 3 days. The individual colonies grown on MMLys platesfrom each transformation were picked and streaked onto MM and MMLysplates. Those colonies that could grow on MMLys plates but not on MMplates were selected as Lys⁻ strains. Single colonies of Lys⁻ strainswere then inoculated into liquid MMLys media at 30° C. and shaken at 250rpm/min for 2 days. The cells were collected by centrifugation, lipidswere extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed increased production of EPA in pZKSL5598transformants. Among the 96 selected Lys-transformants of Y2189 withpZKSL5598, most strains produced between 4-8% EPA of total lipids. Therewere two strains (i.e., #34 and #77, designated herein as “Y2201” and“Y2203”) that produced about 9% and 8.7% EPA of total lipids,respectively.

Example 12 The ω-3 Δ9 Elongase/Δ8 Desaturase Pathway: Generation ofStrains to Produce DHA in Yarrowia lipolytica (Prophetic)

The present Example describes the construction of strains derived fromYarrowia lipolytica ATCC #20362, engineered to produce DHA viaexpression of the ω-3 Δ9 elongase/Δ8 desaturase pathway; thus, analysisof the complete lipid profiles of these DHA-producing strains wouldindicate no GLA co-synthesis in the final DHA-containing oil.

The development of these DHA-producing strains first required creationof strain L116, derived from Yarrowia lipolytica ATCC #20362, capable ofproducing 1.3% EPA relative to the total lipids (FIG. 5). Strain L116required the construction of strain L98 (producing ALA), strain L103(producing increased ALA) and strain L115 (producing about 4% ETA).Additionally, strain L116 required the synthesis and expression of anovel bifunctional Δ5/Δ6 desaturase derived from Danio rerio (GenBankAccession No. BC068224), characterized herein as having only (or strong)ω-3 specificity.

Subsequently, a L116 Ura- strain could be generated by disruption of theUra3 gene in L116 with pKUT16, and then the L116 Ura- strain could betransformed with plasmid pZP2FOEN4, as described above in Example 6,thereby resulting in a transformant strain producing DHA using the ω-3Δ9 elongase/Δ8 desaturase pathway.

Creation Of Lox P::Ura3/HPT::LoxP Integration Constructs and A Cre-SUReplicating Plasmid for Recyclable Selection

The strategy utilized to introduce multiple copies of a Δ15 desaturaseinto Yarrowia lipolytica relied on a recyclable selection marker and asite-specific recombination system (i.e., Cre/Lox). Briefly, the targetgene (i.e., Fusarium moniliforme Δ15 desaturase [SEQ ID NO:51]) wasadjacent to selection markers (e.g., Ura3 and hygromycinphosphotransferase [HPT]) that were flanked by Lox P sites in theintegration construct. Following transformation and selection of thetransformants, the selection marker was excised from the chromosome bythe introduction of a replicating plasmid carrying a sulfonylurearesistance (SU) gene and Cre recombinase gene. Following loss of theselection marker, the Cre plasmid was cured. The cured strain was thusavailable for another round of transformation.

More specifically, plasmid pY72 (FIG. 15A, SEQ ID NO:176) was anintegration construct comprising one copy of the Fusarium moniliformeΔ15 desaturase and a Ura3/HPT selection marker flanked by Lox P sites.Construct pY72 contained the following components:

TABLE 30 Description of Plasmid pY72 (SEQ ID NO: 176) RE Sites AndNucleotides Within SEQ ID NO: 176 Description Of Fragment And ChimericGene Components 6763-7643 881 bp 5′ part of Yarrowia Lip1 gene (GenBankAccession No. Z50020) 9422-10184 763 bp 3′ part of Yarrowia Lip1 gene(GenBank Accession No. Z50020) SwaI/SbfI FBAIN::FmD15:Lip2, comprising:(16-2522) FBAIN: FBAIN promoter (SEQ ID NO: 214) FmD15: Fusariummoniliforme Δ15 desaturase gene (SEQ ID NO: 51) Lip2: Lip2 terminatorsequence from Yarrowia Lip2 gene (GenBank Accession No. AJ012632)2531-2564 LoxP sequence (SEQ ID NO: 382) 2566-4184 Yarrowia Ura3 gene(GenBank Accession No. AJ306421) 4198-5861 TEF::HPT::XPR, comprising:TEF: TEF promoter (GenBank Accession No. AF054508) HPT: Escherichia colihygromycin phosphotransferase coding region, conveying hygromycinresistance (Kaster, K. R., et al., Nucleic Acids Res. 11: 6895-6911(1983)) XPR: ~100 bp of the 3′ region of the Yarrowia Xpr gene (GenBankAccession No. M17741) 5862-5895 LoxP sequence (SEQ ID NO: 382)

Similarly, plasmid pY80 (FIG. 15B, SEQ ID NO:177) was used to create anintegration construct comprising two copies of the Fusarium moniliformeΔ15 desaturase and a Ura3/HPT selection marker flanked by Lox P sites.Using primers 436 and 437 (SEQ ID NOs:383 and 384), PCR was used toamplify the Pac I/Fse I fragment comprising GPD::Fm1::XPR2 from the 8878bp plasmid, pY34 (WO 2005/047480). This Pac I/Fse I fragment was clonedinto Pac I/Fse I-digested vector pY72 by in-fusion cloning (ClontechLaboratories, Inc., Mountain View, Calif.) and transformed into XL-2Ultra competent cells (BRL, Bethesda, Md.). Of the ten positivetransformants identified by miniprep analysis following Pac I/Fse Idigestion, only clones #3 and #4 were correct. One of the correct cloneswas designated “pY80”. Thus, construct pY80 contained the followingcomponents:

TABLE 31 Description of Plasmid pY80 (SEQ ID NO: 177) RE Sites AndNucleotides Within SEQ ID NO: 177 Description Of Fragment And ChimericGene Components PacI/FseI GPD::FmD15:XPR, comprising: (4-2375) GPD: GPDpromoter (SEQ ID NO: 210) FmD15: Fusarium moniliforme Δ15 desaturasegene (SEQ ID NO: 51) XPR: ~100 bp of the 3′ region of the Yarrowia Xprgene (GenBank Accession No. M17741) FseI/SbfI FBAIN::FmD15:Lip2: asdescribed for pY72 (supra) 2385-4891 4900-4933 LoxP sequence (SEQ ID NO:382) 4935-6533 Yarrowia Ura3 gene (GenBank Accession No. AJ306421)6567-8230 TEF::HPT::XPR: as described for pY72 (supra) 8231-8264 LoxPsequence (SEQ ID NO: 382) 8271-9079 809 bp 5′ part of Yarrowia Lip1 gene(GenBank Accession No. Z50020) 11791-12553 763 bp 3′ part of YarrowiaLip1 gene (GenBank Accession No. Z50020)

Construct pY79 (FIG. 15C, SEQ ID NO:178) was a replicating plasmidcarrying a sulfonylurea resistance (SU) gene (i.e., AHAS) and TEF::Crerecombinase gene. Specifically, construct pY79 contained the followingcomponents:

TABLE 32 Description of Plasmid pY79 (SEQ ID NO: 178) RE Sites AndNucleotides Within SEQ ID NO: 178 Description Of Fragment And ChimericGene Components 4329-7315 Yarrowia lipolytica AHAS gene comprising aW497L mutation (SEQ ID NO: 243) 7362-1 TEF::Cre::XPR, comprising: TEF:TEF promoter (GenBank Accession No. AF054508) Cre: Enterobacteria phageP1 Cre gene for recombinase protein (Genbank Accession No. X03453) XPR:~100 bp of the 3′ region of the Yarrowia Xpr gene (GenBank Accession No.M17741)

Generation of Strain L98, Producing ALA

Plasmid pY72 (SEQ ID NO:176) was digested with AscI/SphI, and then usedto transform wild type Yarrowia lipolytica ATCC #20362 using a standardlithium acetate method. Following transformation, the cells were platedonto YPD+Hygromycin (250 μg/mL) plates. After 2 days, 20 transformantswere picked and streaked onto fresh YPD+Hygromycin (250 μg/mL) platesand incubated at 30° C. overnight. The cells were collected bycentrifugation, lipids were extracted, and fatty acid methyl esters wereprepared by trans-esterification, and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed the presence of ALA in pY72 transformants, but not inthe wild type Yarrowia control strain. The best clone produced about 27%ALA of total lipids, and displayed 80% substrate conversion.

The Ura3/HPT markers flanked by the LoxP sites in pY72 were excised fromthe genome by transforming the ATCC #20362/pY72 transformants with pY79(SEQ ID NO:178, carrying the sulfonylurea (SU) resistance marker) andselecting transformants for 3 days on MM+SU (150 μg/mL) plates. TheSU-resistant (SU^(R)) transformants were restreaked on fresh MM+SU (150μg/mL) plates for 1 day and then replica-plated onto YPD+Hygromycin (250μg/mL) plates. All clones (except for clone #1) were sensitive tohygromycin (Hyg^(s)), thus indicating the HPT resistance gene had beensuccessfully excised by the Cre recombinase.

Plasmid pY79 was cured from Hyg^(s) clones #6 and #14 by growing thecells in YPD without selection at 30° C. overnight. Culture (0.1 mL) wasdiluted into 1 mL YPD and used to make a serial dilution, with thehighest dilution being 20,000-fold. Each dilution was then plated onto anew YPD plate and incubated at 30° C. overnight. The plates werereplica-plated on MM+SU (150 μg/mL) plates. All clones were SU-sensitive(SUs), thus indicating that they were successfully cured of pY79. Clone#6-1 was used for additional transformations.

Specifically, using the methodology described above, plasmid pY80 (SEQID NO:177) was digested with AscI/SphI, and then used to transformstrain #6-1. Following selection on YPD+Hygromycin (250 μg/mL) plates,GC analysis of total lipids, transformation with plasmid pY79 (SEQ IDNO:178), identification of SU^(R) and Hyg^(S) clones, and curing ofplasmid pY79, strain #1 was identified. This strain thereby carried 3copies of FmΔ15 and had 96.1% substrate conversion of LA to ALA.

Strain #1 was subjected to transformation with pY80 and subsequentlypY79, as described above. This resulted in creation of strain L98,possessing 5 copies of FmΔ15; however, the Δ15 desaturation in thisstrain was not significantly improved relative to strain #1 (possessing3 copies of FmΔ15), as a result of insufficient substrate (i.e., LA).

Generation of Strain L103, Producing Increased ALA

Plasmid pY86 (FIG. 15D, SEQ ID NO:179) was an integration constructcomprising one copy of the Fusarium moniliforme Δ12 desaturase and aUra3/HPT selection marker flanked by Lox P sites. Specifically, pY86contained the following components:

TABLE 33 Description of Plasmid pY86 (SEQ ID NO: 179) RE Sites AndNucleotides Within SEQ ID NO: 179 Description Of Fragment And ChimericGene Components 3399-4207 809 bp 5′ part of Yarrowia Lip1 gene (GenBankAccession No. Z50020) 6919-7681 763 bp 3′ part of Yarrowia Lip1 gene(GenBank Accession No. Z50020) 28-61 LoxP sequence (SEQ ID NO: 382)63-1681 Yarrowia Ura3 gene (GenBank Accession No. AJ306421) 1695-3358TEF::HPT::XPR, comprising: TEF: TEF promoter (GenBank Accession No.AF054508) HPT: Escherichia coli hygromycin phosphotransferase codingregion, conveying hygromycin resistance (Kaster, K. R., et al., NucleicAcids Res. 11: 6895-6911 (1983)) XPR: ~100 bp of the 3′ region of theYarrowia Xpr gene (GenBank Accession No. M17741) 3359-3392 LoxP sequence(SEQ ID NO: 382) PacI/FseI FBAIN::FmD12::Lip2, comprising: (7690-7)FBAIN: FBAIN promoter (SEQ ID NO: 214) FmD12: Fusarium moniliforme Δ12desaturase gene (SEQ ID NO: 32) Lip2: Lip2 terminator sequence fromYarrowia Lip2 gene (GenBank Accession No. AJ012632)

Using the methodology described above, plasmid pY86 was digested withAscI/SphI, and then used to transform strain L98. Following selection onYPD+Hygromycin (250 μg/mL) plates, GC analysis of total lipids,transformation with plasmid pY79 (SEQ ID NO:178) and identification ofSU^(R) and Hyg^(S) clones, strain L103 was identified. This strainthereby carried 5 copies of FmΔ15, 1 copy of FmΔ12 and was Ura3-.Relative to strain L98, the quantity of 18:1 in strain L103 (as apercent of total fatty acids) was reduced from 42% to about 10%, thequantity of 18:2 in strain L103 (as a percent of total fatty acids) wasincreased from 2% to about 10%, and the quantity of ALA in strain L103(as a percent of total fatty acids) was increased from 22% to 47%.

Generation of Strain L115 to Produce about 4% ETA of Total Lipids

Plasmid pY94 (FIG. 16A, SEQ ID NO:180) was an integration constructcomprising one copy of a Δ8 desaturase, one copy of a Δ9 elongase, and aUra3 selection marker flanked by Lox P sites. This plasmid contained thefollowing components:

TABLE 34 Description of Plasmid pY94 (SEQ ID NO: 180) RE Sites AndNucleotides Within SEQ ID NO: 180 Description Of Fragment And ChimericGene Components PacI/SwaI FBAIN::D8:Pex16, comprising: (1-2587) FBAIN:FBAIN promoter (SEQ ID NO: 214) D8: codon-optimized Δ8 desaturase gene(SEQ ID NO: 81), derived from Euglena gracilis (GenBank Accession No.AF139720) Pex16: Pex16 terminator sequence of Yarrowia Pex16 gene(GenBank Accession No. U75433) 2592-4684 GPAT::D9E::Lip1, comprising:GPAT: GPAT promoter (SEQ ID NO: 216) D9E: codon-optimized Δ9 elongasegene (SEQ ID NO: 71), derived from I. galbana Lip1: Lip1 terminatorsequence of Yarrowia Lip1 gene (GenBank Accession No. Z50020) 4714-4747LoxP sequence (SEQ ID NO: 382) 4761-6378 Yarrowia Ura3 gene (GenBankAccession No. AJ306421) 6380-6413 LoxP sequence (SEQ ID NO: 382)6470-7253 784 bp 5′ part of Yarrowia Ura3 gene (GenBank Accession No.AJ306421) 9965-10480 516 bp 3′ part of Yarrowia Ura3 gene (GenBankAccession No. AJ306421)

Plasmid pY94 was transformed into strain L103, using a standard lithiumacetate method. Following transformation, the cells were plated onto MMplates and maintained for 3 days. Twenty-two colonies were then pickedand streaked onto fresh MM plates and grown at 30° C. overnight. Thecells were collected by centrifugation, lipids were extracted, and fattyacid methyl esters were prepared by trans-esterification, andsubsequently analyzed with a Hewlett-Packard 6890 GC. Clone #8 (hereindesignated as strain L104) possessed the highest Δ9 elongase and Δ8desaturase percent substrate conversions.

The Ura3 marker flanked by the LoxP sites in pY94 was excised from thegenome by transforming log phase cells of strain L104 with 1 μl (˜0.5μg/μl) pY79 (SEQ ID NO:178) and selecting transformants for 4 days onMMU+SU (100 μg/mL) plates. Twelve SU^(R) transformants were restreakedon fresh MM and MMU plates for 2 days. All clones (except one) were URAauxotrophic (i.e., Ura^(S)), thus indicating the Ura3 resistance genehad been successfully excised by the Cre recombinase.

Plasmid pY79 was cured from one URA auxotroph by making 1:10,000 to1:50,000 dilutions in MMU from one-third of a loopful of cells.Dilutions (100 μl/plate) were plated onto YPD plates and incubated at30° C. for 2 days. Eight colonies were picked from a YPD plate andstreaked onto MMU plates and MMU+SU plates and incubated at 30° C. for24 hours. All clones were SU-sensitive (SU^(S)), thus indicating thatthey were successfully cured of pY79. One of these was designated L111and thereby carried 5 copies of FmΔ15, 1 copy of FmΔ12, 1 copy of a Δ8desaturase, 1 copy of a Δ9 elongase and was Ura3-.

Strain L115 (possessing 5 copies of FmΔ15, 1 copy of FmΔ12, 2 copies ofa Δ8 desaturase, 2 copies of a Δ9 elongase and characterized as Ura3-)was created by transforming strain L111 with pY94 (SEQ ID NO:180), usingthe methodology described above. GC analysis showed that strain L115produced about 4% ETA of total lipids (complete lipid profile, infra).

Generation of Strain L116 to Produce about 1.3% EPA of Total Lipids

The Danio rerio desaturase identified as GenBank Accession No. AF309556(Hastings et al., PNAS 98(25):14304-14309 (2001)) was reported to showbifunctional Δ6 and Δ5 desaturase activity in Saccharomyces cerevisiae,with: (1) a distinct preference for ω-3 substrates as compared to ω-6substrates; and, (2) a slightly higher Δ6 desaturase activity relativeto Δ5 desaturase activity.

The Applicants identified GenBank Accession No. BC068224 as a homolog ofGenBank Accession No. AF309556 that differed only by a 1 bp (T) deletionat position 984 of the ORF (resulting in a null mutation) and a 1 bpchange (‘G’ to ‘A’) at position 1171 (resulting in a ‘V’ to ‘M’ aminoacid change).

A mutant protein was then created (identified herein as “Drd6/d5(M)”;SEQ ID NO:18) identical to GenBank Accession No. AF309556 (identifiedherein as “Drd6/d5(V)”; SEQ ID NO:15), with the exception of the V1171Mmutation. Specifically, two overlapping fragments were first amplifiedfrom GenBank Accession No. BC068224 cDNA phagemid using primer pairs 475and 477 (SEQ ID NOs:385 and 386) and 478 and 476 (SEQ ID NOs:387 and388) [wherein primers 477 and 478 carried the “missing T”]. Then, theentire Drd6/d5(M) ORF was amplified using primers 475 and 476 and thetwo overlapping fragments as template. The ORF was placed in areplicating plasmid, containing the following components, and identifiedherein as plasmid “pY91 M” (FIG. 16B):

TABLE 35 Description of Plasmid pY91M (SEQ ID NO: 181) RE Sites AndNucleotides Within SEQ ID NO: 181 Description Of Fragment And ChimericGene Components 2866-4170 ARS18 sequence (GenBank Accession No. A17608)4216-5703 Yarrowia Ura3 gene (GenBank Accession No. AJ306421) SalI/BsiwIFBAIN::DrD6:Pex20, comprising: (5705-8423) FBAIN: FBAIN promoter (SEQ IDNO: 214) DrD6: Drd6/d5(M) gene (SEQ ID NO: 17), derived from Danio rerioΔ5/Δ6 desaturase (GenBank Accession No. BC068224) Pex20: Pex20terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613)

Plasmid pY91V was created from plasmid pY91M by site-specificmutagenesis using a QuikChange® II Site-Directed Mutagenesis Kit,(Stratagene, Catalog #200523) and primers 505 and 506 (SEQ ID NOs:389and 390). pY91V was identical to pY91M, except for a single bp changethat resulted in the M to V amino acid mutation described above.

Plasmids pY91M and pY91V, as well as an empty vector serving as thecontrol, were transformed into log phase cells of strain L115,respectively, using a standard lithium acetate method. Followingtransformation, the cells were plated onto MM plates and maintained for3 days. Colonies were then picked and streaked onto fresh MM plates andgrown at 30° C. overnight. One-third of a loopful of cells from eachclone were inoculated into 3 mL MM and grown in a shaker at 30° C. for24 hrs. Alternatively, cells were grown for 24 hours in MM and thencultured for 3 days in HGM. All cells were harvested and their fattyacid composition was analyzed by GC, as described previously.

The complete lipid profiles of strain L115 (expressing an ω-3 Δ9elongase/Δ8 desaturase pathway as a result of FmΔ15, FmΔ12, Δ8desaturase and Δ9 elongase chimeric genes) transformed with empty vector(control), pY91M and pY91V, are shown below in Table 36. Fatty acids areidentified as 16:0, 16:1, 17:1, 18:0, 18:1 (oleic acid), 18:2 (LA), GLA,20:2 (EDA), DGLA, ARA, ALA, STA, 20:3 (ETrA), ETA and EPA; and thecomposition of each is presented as a % of the total fatty acids. Threeseparate experiments were performed, identified as Experiment No.1, 2and 3 in the column labeled “Exp. No.”. Additionally, the Δ6 and Δ5percent substrate conversions for each strain are reported, with respectto activity utilizing both ω-6 and ω-3 substrates (Table 37).

TABLE 36 Lipid Profile Of Yarrowia lipolytica Strain L115 TransformedWith pY91M And pY91V Exp. Time/ GLA DGLA ARA ALA STA ETA No. StrainMedium 16:0% 16:1% 18:0% 18:1% 18:2% % 20:2% % % % % 20:3% % EPA % 1L115 + 1D MM 16 10 2 8 11 0.0 0 1 0.0 40 3.2 1.1 4.2 1.3 pY91M (clone11) 1 L115 + 1D MM 18 9 5 18 12 0.0 0 1 0.0 31 0.0 0.9 3.9 0.0 control 2L115 + 1D MM/ 14 11 6 26 15 0.5 1 2 0.0 18 2.4 1.0 2.9 0.6 pY91M 3D HGM(clone 11) 2 L115 + 1D MM/ 13 11 6 26 15 0.2 1 2 0.0 20 0.0 1.5 4.1 0.2control 3D HGM 3 L115 + 1D MM 17 8 6 20 15 0.0 0 1 0.0 27 0.0 0.9 3.90.0 pY91V (clone 10) 3 L115 + 1D MM 17 9 3 11 11 0.0 0 0 0.0 38 2.6 1.14.4 1.2 pY91M (clone 11) 3 L115 + 1D MM 17 8 6 21 13 0.0 0 1 0.0 28 0.01.1 4.0 0.0 control * The L115/pY91M transformant identified as clone#11 was designated as Yarrowia lipolytica strain “L116”.

TABLE 37 Percent Substrate Conversion By Drd6/d5M And Drd6/d5V Exp. Δ6Δ6 Δ5 Δ5 No. Strain ω-3/ω-6 (ω-6) (ω-3) (ω-6) (ω-3) 1 L115 + pY91M(clone 11) 4.2 0 7 0 23 1 L115 + control 2.8 0 0 0 0 2 L115 + pY91M(clone 11) 1.3 3 12 0 16 2 L115 + control 1.5 1 0 0 5 3 L115 + pY91V(clone 10) 2.0 0 0 0 0 3 L115 + pY91M (clone 11) 4.4 0 7 21 3 L115 +control 2.5 0 0 0 0

As demonstrated in the results above, expression of Drd6/d5(M) inYarrowia lipolytica (i.e., strain L115+pY91M) did indeed yield abifunctional enzyme having both Δ6 and Δ5 desaturase activities, with ahigher % substrate conversion for Δ5 desaturase activity (i.e., ETA toEPA) than Δ6 desaturase activity (i.e., ALA to STA) and with much higherω-3 substrate preference for both Δ6 and Δ5 desaturase activities.Unexpectedly, Drd6/d5(V) (i.e., strain L115+pY91V) did not show Δ6 or Δ5activity on ω-6 substrates, while Drd6/d5(M) lacked Δ5 activity on ω-6substrate. Thus, Drd6/d5(M) had different characteristics thanDrd6/d5(V). The differences in activity of Drd6/d5(V) from publishedwork are likely to be related to the different host organism in whichthe protein was expressed and/or the origin of the substrate (i.e.,substrate feeding [Hastings et al., supra] or substrate biosynthesis[demonstrated herein]).

To better understand the substrate specificities of Drd6/d5(M) andDrd6/d5(V), the FBAIN::Drd6/d5(M)::Pex20 and FBAIN::Drd6/d5(V)::Pex20chimeric genes were transferred into a Yarrowia replicating plasmid withLEU selection, thereby resulting in creation of plasmids pY102(M) andpY102(V), respectively. These plasmids were then transformed into strainQ-d12D, a Y. lipolytica strain comprising a Δ12 desaturase knockout (WO2004/104167). The transformants were grown for 1 day in MM in thepresence of 0.5 mM of either LA, ALA, ETrA [20:3 (11,14,17)], EDA, DGLAor ETA and the % substrate conversion was tested. Results are shownbelow in Table 38:

TABLE 38 Percent Substrate Conversion By Drd6/d5(M) And Drd6/d5(V) InTransformant Yarrowia Strain Q-d12D Substrate conversion (%) Fatty Δ6 Δ6Δ8 Δ8 Δ5 Δ5 Plasmid Acid (ω-6) (ω-3) (ω-6) (ω-3) (ω-6) (ω-3) pY102(M) LA17 — — — — — pY102(V) LA  4 — — — — — pY102(M) ALA — 24 — — — — pY102(V)ALA —  6 — — — — pY102(M) EDA 17 — 0 — — — pY102(V) EDA  0 — 0 — — —pY102(M) ETrA — 30 — 13 — — pY102(V) ETrA —  9 —  0 — — pY102(M) DGLA —— — — 12 — pY102(V) DGLA — — — —  0 — pY102(M) ETA — — — — — 34 pY102(V)ETA — — — — —  0

The results showed that the novel Drd6/d5(M) desaturase had (as comparedto the published Drd6/d5(V) desaturase): (1) a higher % substrateconversion on all substrates tested; (2) a higher selectivity towardsω-3 fatty acids as compared to ω-6 fatty acids [although there was no Δ5activity in Drd6/d5(V) with either ω-3 or ω-6 substrate]; and, (3) anunexpected Δ8 desaturase activity.

The differences in % substrate conversions between the Q-d12Dtransformants versus L115 transformants were likely the result ofsubstrate feeding. Since Drd6/d5 has been reported to act on an acyl-CoAsubstrate, the desaturase activities can differ as a result of fattyacid feeding or de novo synthesis by the Yarrowia host. Anotherunexpected observation was that Drd6/d5(M) converted ETrA [20:3 (11, 14,17)] into ETA but did not convert EDA [20:2 (11, 14)] into DGLA (i.e.,the protein had Δ8 desaturase activity only on the ω-3 substrate).

It is contemplated that this novel Drd6/d5(M) desaturase hascharacteristics that could provide unique advantages for pathwayengineering when expressed in Yarrowia lipolytica.

Example 13 Generation of Intermediate Strain Y2067U, Producing 14% EPAof Total Lipids

The present Example describes the construction of strain Y2067U, derivedfrom Yarrowia lipolytica ATCC #20362, capable of producing 14% EPArelative to the total lipids (FIG. 5). This strain was engineered toexpress the ω-6 Δ6 desaturase/Δ6 elongase pathway. The effect of M.alpina LPAAT2, DGAT1 and DGAT2 and Y. lipolytica CPT1 geneover-expression was examined in this EPA producing strain based onanalysis of TAG content and/or composition, as described in Examples 17,18, 19 and 24, respectively (infra).

The development of strain Y2067U required the construction of strain M4(producing 8% DGLA and described in Example 6), strain Y2034 (producing10% ARA), strain E (producing 10% EPA), strain EU (producing 10% EPA)and strain Y2067 (producing 15% EPA).

Generation of Y2034 Strain to Produce about 10% ARA of Total Lipids

Construct pDMW232 (FIG. 16C; SEQ ID NO:182) was generated to integratetwo Δ5 chimeric genes into the Leu2 gene of Yarrowia strain M4. PlasmidpDMW232 contained the following components, as described in Table 39:

TABLE 39 Description of Plasmid pDMW232 (SEQ ID NO: 182) RE Sites AndNucleotides Within SEQ ID NO: 182 Description Of Fragment And ChimericGene Components AscI/BsiWI 788 bp 5′ part of Yarrowia Leu2 gene (GenBankAccession (5550-4755) No. AF260230) SphI/PacI 703 bp 3′ part of YarrowiaLeu2 gene (GenBank Accession (8258-8967) No. AF260230) SwaI/BsiWIFBAIN::MAΔ5::Pex20, comprising: (2114-4755) FBAIN: FBAIN promoter (SEQID NO: 214) MAΔ5: Mortierella alpina Δ5 desaturase gene (SEQ ID NO: 6)(GenBank Accession No. AF067654) Pex20: Pex20 terminator sequence ofYarrowia Pex20 gene (GenBank Accession No. AF054613) SwaI/ClaITEF::MAΔ5::Lip1, comprising: (2114-17) TEF: TEF promoter (GenBankAccession No. AF054508) MAΔ5: SEQ ID NO: 6 (supra) Lip1: Lip1 terminatorsequence of Yarrowia Lip1 gene (GenBank Accession No. Z50020) PmeI/ClaIYarrowia Ura3 gene (GenBank Accession No. AJ306421) (5550-4755)

Plasmid pDMW232 was digested with AscI/SphI, and then used to transformstrain M4 according to the General Methods. Following transformation,the cells were plated onto MMLe plates and maintained at 30° C. for 2 to3 days. The individual colonies grown on MMLe plates from eachtransformation were picked and streaked onto MM and MMLe plates. Thosecolonies that could grow on MMLe plates but not on MM plates wereselected as Leu2⁻ strains. Single colonies of Leu2⁻ strains were theninoculated into liquid MMLe media at 30° C. and shaken at 250 rpm/minfor 2 days. The cells were collected by centrifugation, lipids wereextracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed the presence of ARA in pDMW232 transformants, but notin the parental M4 strain. Specifically, among the 48 selected Leu2⁻transformants with pDMW232, there were 34 strains that produced lessthan 5% ARA, 11 strains that produced 6-8% ARA, and 3 strains thatproduced about 10% ARA of total lipids in the engineered Yarrowia. Oneof the strains that produced 10% ARA was named “Y2034”.

Generation of E Strain to Produce about 10% EPA of Total Lipids

Plasmid pZP3L37 (Example 6) was digested with AscI/SphI, and then usedto transform strain Y2034 according to the General Methods. Followingtransformation, the cells were plated onto MM plates and maintained at30° C. for 2 to 3 days. A total of 48 transformants grown on the MMplates were picked and re-streaked onto fresh MM plates. Once grown,these strains were individually inoculated into liquid MM at 30° C. andshaken at 250 rpm/min for 2 days. The cells were collected bycentrifugation, lipids were extracted, and fatty acid methyl esters wereprepared by trans-esterification, and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed the presence of EPA in most of the transformants withpZP3L37, but not in the parental strain (i.e., Y2034). Among the 48selected transformants with pZP3L37, there were 18 strains that producedless than 2% EPA, 14 strains that produced 2-3% EPA, and 1 strain thatproduced about 7% EPA of total lipids in the engineered Yarrowia.

The strain that produced 7% EPA was further analyzed after culturing thestrain using “two-stage growth conditions”, as described in the GeneralMethods (i.e., 48 hrs MM, 72 hrs HGM). GC analyses showed that theengineered strain produced about 10% EPA of total lipids after thetwo-stage growth. The strain was designated as the “E” strain.

Generation of EU Strain to Produce about 10% EPA of Total Lipids withUra- Phenotype

Strain EU (Ura⁻) was created by identifying mutant cells of strain Ethat were 5-FOA resistant. Specifically, one loop of Yarrowia E straincells were inoculated into 3 mL YPD medium and grown at 30° C. withshaking at 250 rpm for 24 hrs. The culture was diluted with YPD to anOD₆₀₀ of 0.4 and then incubated for an additional 4 hrs. The culture wasplated (100 μl/plate) onto MM+FOA plates and maintained at 30° C. for 2to 3 days. A total of 16 FOA resistant colonies were picked and streakedonto MM and MM+FOA selection plates. From these, 10 colonies grew on FOAselection plates but not on MM plates and were selected as potentialUra⁻ strains.

One of these strains was used as host for transformation with pY37/F15,comprising a chimeric GPD::Fusarium moniliforme Δ15::XPR2 gene and aUra3 gene as a selection marker (FIG. 16D; SEQ ID NO:183). After threedays of selection on MM plates, hundreds of colonies had grown on theplates and there was no colony growth of the transformation control thatcarried no plasmid. This experiment confirmed that the 5-FOA resistanthost strain was Ura⁻, and this strain was designated as strain “EU”.

Single colonies of the EU strain were then inoculated into liquid MMUadditionally containing 0.1 g/L uridine and cultured for 2 days at 30°C. with shaking at 250 rpm/min. The cells were collected bycentrifugation, lipids were extracted, and fatty acid methyl esters wereprepared by trans-esterification and subsequently analyzed with aHewlett-Packard 6890 GC. GC analyses showed that the EU strain producedabout 10% EPA of total lipids.

Generation of Y2067 Strain to Produce about 15% EPA of Total Lipids

Plasmid pKO2UF2PE (FIG. 17A; SEQ ID NO:184) was created to integrate acluster containing two chimeric genes (comprising a heterologous Δ12desaturase and a C_(18/20) elongase) and a Ura3 gene into the nativeYarrowia Δ12 desaturase gene of strain EU. Plasmid pKO2UF2PE containedthe following components:

TABLE 40 Description of Plasmid pKO2UF2PE (SEQ ID NO: 184) RE Sites AndNucleotides Within SEQ ID NO: 184 Description Of Fragment And ChimericGene Components AscI/BsiWI 730 bp 5′ part of Yarrowia Δ12 desaturasegene (SEQ ID (3382-2645) NO: 28) SphI/EcoRI 556 bp 3′ part of YarrowiaΔ12 desaturase gene (SEQ ID (6090-6646) NO: 28) SwaI/BsiWI/FBAINm::F.Δ12DS::Pex20, comprising: (1-2645) FBAINm: FBAINm promoter(SEQ ID NO: 215) F.Δ12: Fusarium moniliforme Δ12 desaturase gene (SEQ IDNO: 32) Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene (GenBankAccession No. AF054613) SwaI/PmeI GPAT::EL1S::OCT, comprising: (1-8525)GPAT: GPAT promoter (SEQ ID NO: 216) EL1S: codon-optimized elongase 1gene (SEQ ID NO: 24), derived from Mortierella alpina (GenBank AccessionNo. AX464731) OCT: OCT terminator sequence of Yarrowia OCT gene (GenBankAccession No. X69988) EcoRI/PacI Yarrowia Ura3 gene (GenBank AccessionNo. AJ306421) (6646-8163)

Plasmid pKO2UF2PE was digested with AscI/SphI and then used to transformstrain EU according to the General Methods (although strain EU wasstreaked onto a YPD plate and grown for approximately 36 hr prior tosuspension in transformation buffer [versus 18 hrs]). Followingtransformation, cells were plated onto MM plates and maintained at 30°C. for 2 to 3 days. A total of 72 transformants grown on MM plates werepicked and re-streaked separately onto fresh MM plates. Once grown,these strains were individually inoculated into liquid MM at 30° C. andshaken at 250 rpm/min for 2 days. The cells were collected bycentrifugation, lipids were extracted, and fatty acid methyl esters wereprepared by trans-esterification, and subsequently analyzed with aHewlett-Packard 6890 GC.

GC analyses showed the presence of EPA in almost all of thetransformants with pKO2UF2PE. More specifically, among the 72 selectedtransformants, there were 17 strains that produced 8-9.9% EPA, 27strains that produced 10-10.9% EPA, 16 strains that produced 11-11.9%EPA, and 7 strains that produced 12-12.7% EPA of total lipids in theengineered Yarrowia. The strain that produced 12.7% EPA was furtheranalyzed by using two-stage growth conditions, as described in theGeneral Methods (i.e., 48 hrs MM, 72 hrs HGM). GC analyses showed thatthe engineered strain produced about 15% EPA of total lipids after thetwo-stage growth. The strain was designated as strain “Y2067”.

Generation of Y2067U Strain to Produce about 14% EPA of Total Lipidswith Ura- Phenotype

Plasmid pZKUT16 (Example 6) was digested with SalI/PacI, and then usedto transform Y2067 strain according to the General Methods. Followingtransformation, cells were plated onto MM+5-FOA selection plates andmaintained at 30° C. for 2 to 3 days.

A total of 24 transformants grown on MM+5-FOA plates were picked andre-streaked onto MM plates and MM+5-FOA plates, separately. The strainsthat could grow on MM+5-FOA plates, but not on MM plates, were selectedas Ura- strains. A total of 10 Ura- strains were individually inoculatedinto liquid MMU media at 30° C. and grown with shaking at 250 rpm/minfor 1 day. The cells were collected by centrifugation, lipids wereextracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed the presence of 5 to 7% EPA in all of thetransformants with pZKUT16 after one day growth in MMU media. The strainthat produced 6.2% EPA was further analyzed using the two-stage growthconditions (i.e., 48 hrs MM, 96 hrs HGM). GC analyses showed that theengineered strain produced about 14% EPA of total lipids. The strain wasdesignated as strain “Y2067U”.

The final genotype of this strain with respect to wildtype Yarrowialipolytica ATCC #20362 was as follows: Ura3-, Pox3-, Y.Δ12-,FBA::F.Δ12::Lip2, FBAINm::F. Δ12::Pex20, TEF::Δ6S::Lip1,FBAIN::E1S::Pex20, GPAT::E1S::Oct, TEF::E2S::Xpr, FBAIN::Δ5::Pex20,TEF::Δ5::Lip1, FBAIN::Δ17S::Lip2, FBAINm::Δ17S::Pex16, TEF::Δ17S andTEF::rELO2S::Pex20.

Example 14 Generation of Intermediate Strain Y2107U1, Producing 16% EPAof Total Lipids

The present Example describes the construction of strain Y2107U1,derived from Yarrowia lipolytica ATCC #20362, capable of producingsignificant concentrations of EPA relative to the total lipids (FIG. 5).The affect of M. alpina GPAT gene over-expression was examined in thisEPA producing strain based on analysis of TAG content and/orcomposition, as described in Example 20 (infra).

The development of strain Y2107U1 (producing 16% EPA and possessing aUra- phenotype) herein required the construction of strain M4 (producing8% DGLA and described in Example 6), strain Y2047 (producing 11% ARA anddescribed in Example 6), strain Y2048 (producing 11% EPA and describedin Example 6), strain Y2060 (producing 13% EPA and described in Example6), strain Y2072 (producing 15% EPA and described in Example 6), strainY2072U1 (producing 14% EPA) and Y2089 (producing 18% EPA).

Generation of Y2072U1 Strain to Produce about 14% EPA of Total Lipidswith Ura- Phenotype

The construct pZKUGPI5S (FIG. 17B; SEQ ID NO:187) was created tointegrate a GPAT::I.Δ5S::Pex20 chimeric gene into the Ura3 gene of Y2072strain (Example 6). More specifically, plasmid pZKUGPI5S contained thefollowing components:

TABLE 41 Description of Plasmid pZKUGPI5S (SEQ ID NO: 187) RE Sites AndNucleotides Within SEQ ID NO: 187 Description Of Fragment And ChimericGene Components BsiWI/PacI 721 bp 5′ part of Yarrowia Ura3 gene (GenBankAccession (318-1038) No. AJ306421) SalI/ClaI 724 bp 3′ part of YarrowiaUra3 gene (GenBank Accession (3882-4606) No. AJ306421) ClaI/BsiWIGPAT::I.Δ5S::Pex20, comprising: (4606-318) GPAT: GPAT promoter (SEQ IDNO: 216) I.Δ5S: codon-optimized Δ5 desaturase gene (SEQ ID NO: 10),derived from Isochrysis galbana (WO 2002/ 081668) Pex20: Pex20terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613)

Plasmid pZKUGPI5S was digested with SaII/PacI, and then used totransform strain Y2072 according to the General Methods. Followingtransformation, cells were plated onto MM+5-FOA selection plates andmaintained at 30° C. for 3 to 4 days.

A total of 24 transformants grown on MM+5-FOA plates were picked andre-streaked onto MM plates and MM+5-FOA plates, separately. Thosestrains that could grow on MM+5-FOA plates, but not on MM plates, wereselected as Ura- strains. Each of these 24 Ura- strains wereindividually inoculated into liquid MMU and grown at 30° C. with shakingat 250 rpm/min for 2 days. The cells were collected by centrifugation,lipids were extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed that there were 8 strains that produced 7.3-8.9% EPA,14 strains that produced 9-9.9% EPA, 1 strain that produced 10.5% EPA(i.e., #1) and 1 strain that produced 10.7% EPA (i.e., #23) of totallipids after two day growth in MMU. Strains #1 and #23 were furtheranalyzed using two-stage growth conditions (i.e., 48 hrs MM, 96 hrsHGM). GC analyses showed that these two strains produced about 14% EPAof total lipids after the two-stage growth. Strain #1 was designated asstrain “Y2072U1”.

Generation of Y2089 Strain to Produce about 18% EPA of Total Lipids

Construct pDMW302T16 (FIG. 17C; SEQ ID NO:186) was created to integratea cluster of four chimeric genes (comprising a C_(16/18) elongase, aC_(18/20) elongase, a Δ6 desaturase and a Δ12 desaturase) and a Ura3gene into the Yarrowia lipase1 gene site of Y2072U1 strain. PlasmidpDMW302T16 contained the following components:

TABLE 42 Description of Plasmid pDMW302T16 (SEQ ID NO: 186) RE Sites AndNucleotides Within SEQ Description Of ID NO: 186 Fragment And ChimericGene Components BsiWI/AscI 817 bp 5′ part of Yarrowia lipase1 gene(GenBank (1-817) Accession No. Z50020) SphI/PacI 769 bp 3′ part ofYarrowia lipase1 gene (GenBank 3525-4294 Accession No. Z50020)EcoRI/BsiWI TEF::rELO2S::Pex20, comprising: (13328-1) TEF: TEF promoter(GenBank Accession No. AF054508) rELO2S: codon-optimized rELO2 elongasegene (SEQ ID NO: 85), derived from rat (GenBank Accession No. AB071986)Pex20: Pex20 terminator sequence of Yarrowia Pex20 gene (GenBankAccession No. AF054613) BglII/EcoRI FBAIN::D6S::Lip1, comprising:(10599-13306) FBAIN: FBAIN promoter (SEQ ID NO: 214) Δ6S:codon-optimized Δ6 desaturase gene (SEQ ID NO: 3), derived fromMortierella alpina (GenBank Accession No. AF465281) Lip1: Lip1terminator sequence from Yarrowia Lip1 gene (GenBank Accession No.Z50020) ClaI/PmeI GPDIN::EL1S::Lip2, comprising: (8078-10555) GPDIN:GPDIN promoter (SEQ ID NO: 211) EL1S: codon-optimized elongase 1 gene(SEQ ID NO: 24), derived from Mortierella alpina (GenBank Accession No.AX464731) Lip2: Lip2 terminator of Yarrowia lipase2 gene (GenBankAccession No. AJ012632) EcoRI/ClaI Yarrowia Ura 3 gene (Gene BankAccession No. (6450-8078) AJ306421) PacI/EcoRI TEF:: F.Δ12::Pex16,comprising: (4294-6450) TEF: TEF promoter (GenBank Accession No.AF054508) F.Δ12: Fusarium moniliforme Δ12 desaturase gene (SEQ ID NO:32) Pex16: Pex16 terminator of Yarrowia Pex16 gene (GenBank AccessionNo. U75433)

Plasmid pDMW302T16 was digested with SphI/AscI, and then used totransform strain Y2072U1 according to the General Methods. Followingtransformation, cells were plated onto MM plates and maintained at 30°C. for 3 to 4 days.

A total of 48 transformants grown on MM plates were picked andre-streaked onto fresh MM plates. Once grown, these strains wereindividually inoculated into liquid MM and grown at 30° C. with shakingat 250 rpm/min for 2 days. The cells were collected by centrifugation,lipids were extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed that EPA was produced in almost all transformants ofY2072U1 with pDMW302T16 after two-day growth in MM media. Among the 48selected transformants, there were 27 strains that produced less than10% EPA, 14 strains that produced 10-12.9% EPA and 5 strains thatproduced 13-13.9% EPA. Strain #34 (producing 13.9% EPA) was selected forfurther analysis using the two-stage growth procedure (i.e., 48 hrs MM,96 hrs HGM). GC analyses showed that strain #34 produced about 18% EPAof total lipids. Strain #34 was designated as strain “Y2089”.

The genotype of strain Y2089 with respect to wildtype Yarrowialipolytica ATCC #20362 was as follows: Pox3-, LIP1-, Y.Δ12-,FBA::F.Δ12::Lip2, TEF::F. Δ12::Pex16, FBAIN::MΔ12::Pex20,TEF::Δ6S::Lip1, FBAIN::Δ6S::Lip1, FBAIN::E1S::Pex20, GPAT::E1S::Oct,GPDIN::E1S::Lip2, TEF::E2S::Xpr, FBAIN::MAΔ5::Pex20, TEF::MAΔ5::Lip1,TEF::HΔ5S::Pex16, TEF::IΔ5S::Pex20, GPAT::IΔ5S::Pex20,FBAIN::Δ17S::Lip2, FBAINm::Δ17S::Pex16, TEF::Δ17S::Pex16 and 2×TEF::rELO2S::Pex20.

Generation of Y2107U1 Strain to Produce about 16% EPA of Total Lipidswith Ura- Phenotype

Construct pZKUGPE1S (SEQ ID NO:187) was created to integrate aGPAT::EL1S::Pex20 chimeric gene into the Ura3 gene of strain Y2089. Morespecifically, plasmid pZKUGPE1S contained the following components:

TABLE 43 Description of Plasmid pZKUGPE1S (SEQ ID NO: 187) RE Sites AndNucleotides Within SEQ ID NO: 187 Description Of Fragment And ChimericGene Components BsiWI/PacI 721 bp 5′ part of Yarrowia Ura3 gene (GenBankAccession (318-1038) No. AJ306421) SalI/ClaI 724 bp 3′ part of YarrowiaUra3 gene (GenBank Accession (3882-4606) No. AJ306421) ClaI/BsiWIGPAT::E1S::Pex20, comprising: (4606-318) GPAT: GPAT promoter (SEQ ID NO:216) EL1S: codon-optimized elongase 1 gene (SEQ ID NO: 24), derived fromMortierella alpina (GenBank Accession No. AX464731) Pex20: Pex20terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613)

Plasmid pZKUGPE1S was digested with PstI/PacI, and then used totransform strain Y2089 according to the General Methods. Followingtransformation, cells were plated onto MM+5-FOA selection plates andmaintained at 30° C. for 3 to 4 days.

A total of 8 transformants grown on MM+5-FOA plates were picked andre-streaked onto MM plates and MM+5-FOA plates, separately. Thosestrains that could grow on MM+5-FOA plates, but not on MM plates, wereselected as Ura- strains. Each of these 8 Ura- strains were individuallyinoculated into liquid MMU and grown at 30° C. with shaking at 250rpm/min for 2 days. The cells were collected by centrifugation, lipidswere extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed that there were 6 strains that produced 6.6-8.7% EPAand 2 strains that produced 9.4-10% EPA (i.e., #4 and #5) of totallipids after two day growth in MMU. Strains #4 and #5 were furtheranalyzed using the two-stage growth conditions (i.e., 48 hrs MM, 96 hrsHGM). GC analyses showed that these two strains produced about 16% EPAof total lipids after the two-stage growth. Strain #4 was designated asstrain “Y2107U1” and strain #5 was designated as strain “Y2107U2”.

Example 15 Generation of Intermediate Strain MU, Producing 9-12% EPA ofTotal Lipids

The present Example describes the construction of strain MU, derivedfrom Yarrowia lipolytica ATCC #20362, capable of producing significantconcentrations of EPA relative to the total lipids (FIG. 5). The affectof various native Y. lipolytica acyltransferase knockouts were examinedin this EPA producing strain based on analysis of TAG content and/orcomposition, as described in Example 27 (infra).

The development of strain MU (producing 9-12% EPA herein) required theconstruction of strain M4 (producing 8% DGLA and described in Example6), strain Y2034 (producing 10% ARA and described in Example 13), strainE (producing 10% EPA and described in Example 13), strain EU (producing10% EPA and described in Example 13) and strain M26 (producing 14% EPA).

Generation of M26 Strain to Produce about 14% EPA of Total Lipids

Construct pKO2UM26E (SEQ ID NO:188; FIG. 17D) was used to integrate acluster of three chimeric genes (comprising a C_(18/20) elongase, a Δ6desaturase and a Δ12 desaturase) and a Ura3 gene into the Yarrowia Δ12desaturase gene site of EU strain (Example 13). Plasmid pKO2UM26Econtained the following components:

TABLE 44 Description of Plasmid pKO2UM26E (SEQ ID NO: 188) RE Sites AndNucleotides Within SEQ ID Description Of NO: 188 Fragment And ChimericGene Components HindIII/AscI 728 bp 5′ part of Yarrowia Δ12 desaturasegene (SEQ ID (1-728) NO: 28) SphI/EcoRI 556 bp 3′ part of Yarrowia Δ12desaturase gene (SEQ ID (3436-3992) NO: 28) BsiWI/HindIIIGPAT::EL1S::XPR, comprising: (11095-1) GPAT: GPAT promoter (SEQ ID NO:216) EL1S: codon-optimized elongase 1 gene (SEQ ID NO: 24), derived fromMortierella alpina (GenBank Accession No. AX464731) XPR: ~100 bp of the3′ region of the Yarrowia Xpr gene (GenBank Accession No. M17741)BglII/BsiWI FBAIN::M.Δ12::Pex20, comprising: (8578-11095) FBAIN: FBAINpromoter (SEQ ID NO: 214) M.Δ12: Mortieralla isabellina Δ12 desaturasegene (GenBank Accession No. AF417245; SEQ ID NO: 30) Pex20: Pex20terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613) SalI/PacI Yarrowia Ura3 gene (GenBank Accession No.(6704-8202) AJ306421) EcoRI/SalI FBAIN::M.Δ6B::Pex20, comprising:(3992-6704) TEF: TEF promoter (GenBank Accession No. AF054508) M.Δ6B:Mortieralla alpina Δ6 desaturase gene “B” (GenBank Accession No.AB070555; SEQ ID NO: 4) Pex20: Pex20 terminator sequence of YarrowiaPex20 gene (GenBank Accession No. AF054613)

The plasmid pKO2UM26E was digested with SphI/AscI, and then used totransform EU strain (Example 13) according to the General Methods.Following transformation, cells were plated onto MM plates andmaintained at 30° C. for 2 to 3 days.

A total of 48 transformants grown on MM plates were picked andre-streaked onto fresh MM plates. Once grown, these strains wereindividually inoculated into liquid MM at 30° C. and grown with shakingat 250 rpm/min for 1 day. The cells were collected by centrifugation,lipids were extracted, and fatty acid methyl esters were prepared bytrans-esterification, and subsequently analyzed with a Hewlett-Packard6890 GC.

GC analyses showed that EPA was produced in almost all transformantswith pKO2UM26E after one-day growth in MM media. Among the 48 selectedtransformants, 5 strains produced less than 4% EPA, 23 strains produced4-5.9% EPA, 9 strains produced 6-6.9% EPA and 11 strains produced 7-8.2%EPA of total lipids in the engineered Yarrowia. The strain that produced8.2% EPA was selected for further analysis using the two-stage growthprocedure (i.e., 48 hrs MM, 96 hrs HGM). GC analyses showed that theengineered strain produced about 14% EPA of total lipids. The strain wasdesignated as strain “M26”.

The genotype of the M26 strain with respect to wildtype Yarrowialipolytica ATCC #20362 was as follows: Pox3-, Y.Δ12-, FBA::F.Δ12::Lip2,FBAIN::MΔ12::Pex20, TEF::Δ6S::Lip1, FBAIN::Δ6B::Pex20,FBAIN::E1S::Pex20, GPAT::E1S::Xpr, TEF::E2S::Xpr, FBAIN::MAΔ5::Pex20,TEF::MAΔ5::Lip1, TEF::HΔ5S::Pex16, FBAIN::Δ17S::Lip2,FBAINm::Δ17S::Pex16, TEF::Δ17S::Pex16 and TEF::rELO2S::Pex20.

Generation of MU Strain to Produce about 14% EPA of Total Lipids

Strain MU was a Ura auxotroph of strain M26. This strain was made bytransforming strain M26 with 5 μg of plasmid pZKUM (SEQ ID NO:189) thathad been digested with PacI and HincII. Transformation was performedusing the Frozen-EZ Yeast Transformation kit (Zymo Research Corporation,Orange, Calif.) and transformants were selected by plating 100 μl of thetransformed cell mix on an agar plate with the following medium: 6.7 g/Lyeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.), 20 g/Ldextrose, 50 mg/L uracil and 800 mg/L FOA. After 7 days, small coloniesappeared that were plated on MM and MMU agar plates. All were URAauxotrophs. One of the strains was designated “MU”.

Example 16 Preparation of Mortierella alpina Genomic DNA and cDNA

The present Example describes the preparation of genomic DNA and cDNAfrom Mortierella alpina (ATCC #16266). This enabled isolation of the M.alpina LPAAT2, DGAT1, DGAT2, GPAT and ELO3, as described in Examples 17,18, 19, 20 and 21, respectively.

Preparation of Genomic DNA from Mortierella alpina

Genomic DNA was isolated from Mortierella alpina (ATCC #16266) using aQiaPrep Spin Miniprep Kit (Qiagen, Catalog #627106). Cells grown on aYPD agar plate (2% Bacto-yeast extract, 3% Bacto-peptone, 2% glucose,2.5% bacto-agar) were scraped off and resuspended in 1.2 mL of kitbuffer P1. The resuspended cells were placed in two 2.0 mL screw captubes, each containing 0.6 mL glass beads (0.5 mm diameter). The cellswere homogenized at the HOMOGENIZE setting on a Biospec (Bartlesville,Okla.) mini bead beater for 2 min. The tubes were then centrifuged at14,000 rpm in an Eppendorf microfuge for 2 min. The supernatant (0.75mL) was transferred to three 1.5 mL microfuge tubes. Equal volumes ofkit buffer P2 were added to each tube. After mixing the tubes byinversion three times, 0.35 mL of buffer N3 was added to each tube. Thecontents of each tube were again mixed by inversion for a total of fivetimes. The mixture was centrifuged at 14,000 rpm in an Eppendorfmicrofuge for 5 min. The supernatant from each tube was transferredindividually into 3 separate kit spin columns. The columns were thensubjected to the following steps: centrifugation (1 min at 14,000 rpm),wash once with buffer PE, centrifugation (1 min at 14,000 rpm), and thena final centrifugation (1 min at 14,000 rpm). Buffer EB (50 μl) wasadded to each column and let stand for 1 min. The genomic DNA was theneluted by centrifugation at 14,000 rpm for 1 min.

Preparation of cDNA from Mortierella alpina

cDNA of Mortierella alpina was prepared using the BD-Clontech CreatorSmart® cDNA library kit (Mississauga, ON, Canada), according to themanufacturer's protocol.

Specifically, M. alpina strain ATCC #16266 was grown in 60 mL YPD medium(2% Bacto-yeast extract, 3% Bactor-peptone, 2% glucose) for 3 days at23° C. Cells were pelleted by centrifugation at 3750 rpm in a BeckmanGH3.8 rotor for 10 min and resuspended in 6× 0.6 mL Trizole reagent(Invitrogen). Resuspended cells were transferred to six 2 mL screw captubes each containing 0.6 mL of 0.5 mm glass beads. The cells werehomogenized at the HOMOGENIZE setting on a Biospec mini bead beater for2 min. The tubes were briefly spun to settle the beads. Liquid wastransferred to 4 fresh 1.5 mL microfuge tubes and 0.2 mLchloroform/isoamyl alcohol (24:1) was added to each tube. The tubes wereshaken by hand for 1 min and let stand for 3 min. The tubes were thenspun at 14,000 rpm for 10 min at 4° C. The upper layer was transferredto 4 new tubes. Isopropyl alcohol (0.5 mL) was added to each tube. Tubeswere incubated at room temperature for 15 min, followed bycentrifugation at 14,000 rpm and 4° C. for 10 min. The pellets werewashed with 1 mL each of 75% ethanol, made with RNase-free water andair-dried. The total RNA sample was then redissolved in 500 μl of water,and the amount of RNA was measured by A260 nm using 1:50 diluted RNAsample. A total of 3.14 mg RNA was obtained.

This total RNA sample was further purified with the Qiagen RNeasy totalRNA Midi kit following the manufacturer's protocol. Thus, the total RNAsample was diluted to 2 mL and mixed with 8 mL of buffer RLT with 80 μlof β-mercaptoethanol and 5.6 mL 100% ethanol. The sample was dividedinto 4 portions and loaded onto 4 RNeasy midid columns. The columns werethen centrifuged for 5 min at 4500×g. To wash the columns, 2 mL ofbuffer RPE was loaded and the columns centrifuged for 2 min at 4500×g.The washing step was repeated once, except that the centrifugation timewas extended to 5 min. Total RNA was eluted by applying 250 μl of RNasefree water to each column, waiting for 1 min and centrifuging at 4500×gfor 3 min.

PolyA(+)RNA was then isolated from the above total RNA sample, followingthe protocol of Amersham Biosciences' mRNA Purification Kit. Briefly, 2oligo-dT-cellulose columns were used. The columns were washed twice with1 mL each of high salt buffer. The total RNA sample from the previousstep was diluted to 2 mL total volume and adjusted to 10 mM Tris/HCl, pH8.0, 1 mM EDTA. The sample was heated at 65° C. for 5 min, then placedon ice. Sample buffer (0.4 mL) was added and the sample was then loadedonto the two oligo-dT-cellulose columns under gravity feed. The columnswere centrifuged at 350×g for 2 min, washed 2× with 0.25 mL each of highsalt buffer, each time followed by centrifugation at 350×g for 2 min.The columns were further washed 3 times with low salt buffer, followingthe same centrifugation routine. Poly(A)+RNA was eluted by washing thecolumn 4 times with 0.25 mL each of elution buffer preheated to 65° C.,followed by the same centrifugation procedure. The entire purificationprocess was repeated once. Purified poly(A)+RNA was obtained with aconcentration of 30.4 ng/μl.

cDNA was generated, using the LD-PCR method specified by BD-Clontech and0.1 μg of polyA(+)RNA sample. Specifically, for 1^(st) strand cDNAsynthesis, 3 μl of the poly(A)+RNA sample was mixed with 1 μl of SMARTIV oligo nucleotide (SEQ ID NO:391) and 1 μl of CDSIII/3′ PCR primer(SEQ ID NO:392). The mixture was heated at 72° C. for 2 min and cooledon ice for 2 min. To the tube was added the following: 2 μl first strandbuffer, 1 μl 20 mM DTT, 1 μl 10 mM dNTP mix and 1 μl Powerscript reversetranscriptase. The mixture was incubated at 42° C. for 1 hr and cooledon ice.

The 1^(st) strand cDNA synthesis mixture was used as template for thePCR reaction. Specifically, the reaction mixture contained thefollowing: 2 μl of the 1^(st) strand cDNA mixture, 2 μl 5′-PCR primer(SEQ ID NO:393), 2 μl CDSIII/3′-PCR primer (SEQ ID NO:392), 80 μl water,10 μl 10×Advantage 2 PCR buffer, 2 μl 50× dNTP mix and 2 μl 50×Advantage 2 polymerase mix. The thermocycler conditions were set for 95°C for 20 sec, followed by 14-20 cycles of 95° C. for 5 sec and 68° C.for 6 min on a GenAmp 9600 instrument. PCR product was quantitated byagarose gel electrophoresis and ethidium bromide staining.

Seventy-five μl of the above PCR products (cDNA) were mixed with 3 μl of20 μg/μl proteinase K supplied with the kit. The mixture was incubatedat 45° C. for 20 min, then 75 μl of water was added and the mixture wasextracted with 150 μl phenol:chloroform:isoamyl alcohol mixture(25:24:1). The aqueous phase was further extracted with 150 μlchloroform:isoamyl alcohol (25:1). The aqueous phase was then mixed with15 μl of 3 M sodium acetate, 2 μl of 20 μg/μl glycogen and 400 μl of100% ethanol. The mixture was immediately centrifuged at roomtemperature for 20 min at 14000 rpm in a microfuge. The pellet waswashed once with 150 μl of 80% ethanol, air dried and dissolved in 79 μlof water.

Dissolved cDNA was subsequently digested with SfiI (79 μl of the cDNAwas mixed with 10 μl of 10× SfiI buffer, 10 μl of SfiI enzyme and 1 μlof 100×BSA and the mixture was incubated at 50° C. for 2 hrs). Xylenecyanol dye (2 μl of 1%) was added. The mixture was then fractionated onthe Chroma Spin-400 column provided with the kit, following themanufacturer's procedure exactly. Fractions collected from the columnwere analyzed by agarose gel electrophoresis. The first three fractionscontaining cDNA were pooled and cDNA precipitated with ethanol. Theprecipitated cDNA was redissolved in 7 μl of water, and ligated intokit-supplied pDNR-LIB.

Library Sequencing

The ligation products were used to transform E. coli XL-1 Blueelectroporation competent cells (Stratagene). An estimated total of2×10⁶ colonies was obtained. Sequencing of the cDNA library was carriedout by Agencourt Bioscience Corporation (Beverly, Mass.), using an M13forward primer (SEQ ID NO:394).

Example 17 Mortierella alpina LPAAT2 Expression Increases Percent PUFAs

The present Example describes increased EPA biosynthesis andaccumulation in Yarrowia lipolytica strain Y2067U (Example 13) that wastransformed to co-express the M. alpina LPAAT2 (SEQ ID NOs:110 and 111).It is contemplated that a Y. lipolytica host strain engineered toproduce DHA via either the Δ6 desaturase/Δ6 elongase pathway or the Δ9elongase/Δ8 desaturase pathway could demonstrate increased DHAbiosynthesis and accumulation, if the M. alpina LPAAT2 was similarlyco-expressed therein (e.g., in strain Y3000).

The M. alpina LPAAT2 ORF was cloned as follows. Primers MLPAT-F andMLPAT-R (SEQ ID NOs:395 and 396) were used to amplify the LPAAT2 ORFfrom the cDNA of M. alpina (Example 16) by PCR. The reaction mixturecontained 1 μl of the cDNA, 1 μl each of the primers, 22 μl water and 25μl ExTaq premix 2×Taq PCR solution (TaKaRa Bio Inc., Otsu, Shiga,520-2193, Japan). Amplification was carried out as follows: initialdenaturation at 94° C. for 150 sec, followed by 30 cycles ofdenaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec andelongation at 72° C. for 90 sec. A final elongation cycle at 72° C. for10 min was carried out, followed by reaction termination at 4° C. A ˜950bp DNA fragment was obtained from the PCR reaction. It was purifiedusing a Qiagen (Valencia, Calif.) PCR purification kit according to themanufacturer's protocol. The purified PCR product was digested with NcoIand NotI, and cloned into Nco I-Not I cut pZUF17 vector (SEQ ID NO:162;FIG. 9B), such that the gene was under the control of the Y. lipolyticaFBAIN promoter and the PEX20-3′ terminator region in theauto-replicating vector for expression in Y. lipolytica. Correcttransformants were confirmed by restriction analysis of miniprep DNA andthe resultant plasmid was designated as “pMLPAT-17” (SEQ ID NO:190).

To integrate the M. alpina LPMT2 into the genome of Yarrowia lipolytica,plasmid pMLPAT-Int was created. Primers LPAT-Re-5-1 and LPAT-Re-5-2 (SEQID NOs:397 and 398) were used to amplify a 1129 bp DNA fragment,YLPAT-5′ (SEQ ID NO:399), containing a 1103 bp fragment of Y. lipolyticagenome immediately upstream of the AUG of the Y. lipolytica LPAAT1 (SEQID NO:112). The reaction mixture contained 1 μl of Y. lipolytica genomicDNA, 1 μl each of the primers, 22 μl water and 25 μl ExTaq premix 2×TaqPCR solution (TaKaRa). Amplification was carried out as described above.A ˜1130 bp DNA fragment was obtained from the PCR reaction. It waspurified using Qiagen's PCR purification kit according to themanufacturer's protocol. The purified PCR product was digested with SalIand ClaI, and cloned into SaII-ClaI cut pBluescript SK (−) vector,resulting in plasmid “pYLPAT-5′”.

Primers LPAT-Re-3-1 and LPAT-Re-3-2 (SEQ ID NOs:400 and 401) were thenused to amplify a 938 bp fragment, YLPAT-3′ (SEQ ID NO:402), containinga 903 bp fragment of Y. lipolytica genome immediately after the stopcodon of Y. lipolytica LPAAT1, using the same conditions as above. Thepurified PCR product was digested with ClaI and XhoI, and cloned intoClaI-XhoI digested pYLPAT-5′. Correct transformants were confirmed byminiprep analysis and the resultant plasmid was designated“pYLPAT-5′-3′”.

pMLPAT-17 (SEQ ID NO:190) was then digested with ClaI and NotI, and a˜3.5 kb fragment containing the Y. lipolytica URA3 gene, the Y.lipolytica FBAIN promoter and the M. alpina LPAAT2 gene was isolatedusing a Qiagen QiaexII gel purification kit according to themanufacturer's protocol. This fragment was cloned into ClaI-NotIdigested pYLPAT-5′-3′. Correct transformants were confirmed by miniprepand restriction analysis. The resulting plasmid was named “pMLPAT-Int”(SEQ ID NO:191).

“Control” vector pZUF-MOD-1 (SEQ ID NO:192; FIG. 18A) was prepared asfollows. First, primers pzuf-mod1 and pzuf-mod2 (SEQ ID NOs:403 and 404)were used to amplify a 252 bp “stuffer” DNA fragment using pDNR-LIB(ClonTech, Palo Alto, Calif.) as template. The amplified fragment waspurified with a Qiagen QiaQuick PCR purification kit, digested with NcoIand NotI using standard conditions, and then purified again with aQiaQuick PCR purification kit. This fragment was ligated into similarlydigested NcoI-/NotI/-cut pZUF17 vector (SEQ ID NO:162; FIG. 9B) and theresulting ligation mixture was used to transform E. coli Top10 cells(Invitrogen). Plasmid DNA was purified from 4 resulting colonies, usinga Qiagen QiaPrep Spin Miniprep kit. The purified plasmids were digestedwith NcoI and NotI to confirm the presence of the ˜250 bp fragment. Theresulting plasmid was named “pZUF-MOD-1” (SEQ ID NO:192).

Y. lipolytica strain Y2067U (from Example 13, producing 14% EPA of totallipids) was transformed with plasmid pMLPAT-17, plasmid pZUF-MOD-1(control) and SpeI/XbaI digested plasmid pMLPAT-Int, individually,according to the General Methods. Transformants were grown for 2 days insynthetic MM supplemented with amino acids, followed by 4 days in HGM.The fatty acid profile of two transformants containing pZUF-MOD-1, twotransformants containing pMLPAT-17, and two transformants havingpMLPAT-Int integrated into the genome are shown below in the Table,based on GC analysis (as described in the General Methods). Fatty acidsare identified as 18:0, 18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA,ETA and EPA; and the composition of each is presented as a % of thetotal fatty acids.

TABLE 45 Lipid Composition In Yarrowia Strain Y2067U Engineered ToOverexpress M. alpina LPAAT2 Total Fatty Acids Strain 18:0 18:1 18:2 GLADGLA ARA ETA EPA Y2067U + pZUF-MOD-1 #1 1.1 4.7 10.9 19.4 6.3 0.9 3.913.8 Y2067U + pZUF-MOD-1 #2 0.9 4.4 9.5 19.3 6.6 0.9 4.0 14.1 Y2067U +pMLPAT-17 #1 1.0 4.4 9.8 18.6 5.9 0.8 3.4 15.5 Y2067U + pMLPAT-17 #2 0.73.5 8.4 16.7 6.2 1.0 2.9 16.0 Y2067U + pMLPAT-Int #1 1.9 4.9 13.9 21.14.8 1.1 2.7 16.6 Y2067U + pMLPAT-Int #2 1.7 4.2 12.1 21.3 5.2 1.2 2.917.3

As demonstrated above, expression of the M. alpina LPAAT2 from pMLPAT-17increased the % EPA from ˜14% in the “control” strains to 15.5-16%. Anadditional increase in EPA to 16.6-17.3% was achieved when M. alpinaLPAAT2 was integrated into the genome with pMLPAT-Int. Further increasewould be expected, if the native Yarrowia lipolytica LPAAT1 (SEQ IDNOs:112 and 113) and/or LPAAT2 (SEQ ID NOs:115 and 116) were knocked-outin e.g., strain Y2067U+pMLPAT-Int.

Example 18 Mortierella alpina DGAT1 Expression Increases Percent PUFAs

The present Example describes increased EPA biosynthesis andaccumulation in Yarrowia lipolytica strain Y2067U (Example 13) that wastransformed to co-express the M. alpina DGAT1 cDNA (SEQ ID NO:124). Itis contemplated that a Y. lipolytica host strain engineered to produceDHA via either the Δ6 desaturase/Δ6 elongase pathway or the Δ9elongase/Δ8 desaturase pathway could demonstrate increased DHAbiosynthesis and accumulation, if the M. alpina DGAT1 was similarlyco-expressed therein (e.g., in strain Y3000).

The M. alpina DGAT1 ORF was cloned as follows. First, to aid the cloningof the cDNA, the sequence of the second codon of the DGAT1 was changedfrom ‘ACA’ to ‘GCA’, resulting in an amino acid change of threonine toalanine. This was accomplished by amplifying the complete coding regionof the M. alpina DGAT1 ORF with primers MACAT-F1 and MACAT-R (SEQ IDNOs:405 and 406). Specifically, the PCR reaction mixture contained 1 μleach of a 20 μM solution of primers MACAT-F1 and MACAT-R, 1 μl of M.alpina cDNA (supra, Example 16), 22 μl water and 25 μI ExTaq premix2×Taq PCR solution (TaKaRa Bio Inc., Otsu, Shiga, 520-2193, Japan).Amplification was carried out as follows: initial denaturation at 94° C.for 150 sec, followed by 30 cycles of denaturation at 94° C. for 30 sec,annealing at 55° C. for 30 sec, and elongation at 72° C. for 90 sec. Afinal elongation cycle at 72° C. for 10 min was carried out, followed byreaction termination at 4° C. A ˜1600 bp DNA fragment was obtained fromthe PCR reaction. It was purified using Qiagen's PCR purification kitaccording to the manufacturer's protocol.

The M. alpina DGAT1 ORF was to be inserted into Nco I- and NotI-digested plasmid pZUF17 (SEQ ID NO:162; FIG. 9B), such that the ORFwas cloned under the control of the FBAIN promoter and the PEX20-3′terminator region. However, since the DGAT1 ORF contained an internalNcoI site, it was necessary to perform two separate restriction enzymedigestions for cloning. First, ˜2 μg of the purified PCR product wasdigested with BamHI and Nco I. The reaction mixture contained 20 U ofeach enzyme (Promega) and 6 μl of restriction buffer D in a total volumeof 60 μl. The mixture was incubated for 2 hrs at 37° C. A ˜320 bpfragment was separated by agarose gel electrophoresis and purified usinga Qiagen Qiaex II gel purification kit. Separately, ˜2 μg of thepurified PCR product was digested with BamHI and Not I using identicalreaction conditions to those above, except Nco I was replaced by Not I.A ˜1280 bp fragment was isolated and purified as above. Finally, ˜3 μgof pZUF17 was digested with Nco I and Not I and purified as describedabove, generating a ˜7 kB fragment.

The ˜7 kB Nco I/Not I pZUF17 fragment, the ˜320 bp Nco I/BamHI DGAT1fragment and the ˜1280 bp BamHI/Not I DGAT1 fragment were ligatedtogether in a three-way ligation incubated at room temperatureovernight. The ligation mixture contained 100 ng of the 7 kB fragmentand 200 ng each of the 320 bp and 1280 bp fragments, 2 μl ligase buffer,and 2 U T4 DNA ligase (Promega) in a total volume of 20 μl. The ligationproducts were used to transform E. coli Top10 chemical competent cells(Invitrogen) according to the manufacturer's protocol.

Individual colonies (12 total) from the transformation were used toinoculate cultures for miniprep analysis. Restriction mapping andsequencing showed that 5 out of the 12 colonies harbored the desiredplasmid, which was named “pMDGAT1-17” (FIG. 18B; SEQ ID NO:193).

Y. lipolytica strain Y2067U (from Example 13) was transformed withpMDGAT1-17 and pZUF-MOD-1 (supra, Example 17), respectively, accordingto the General Methods. Transformants were grown for 2 days in syntheticMM supplemented with amino acids, followed by 4 days in HGM. The fattyacid profile of two transformants containing pMDGAT1-17 and twotransformants containing pZUF-MOD-1 are shown below in Table 46, basedon GC analysis (as described in the General Methods). Fatty acids areidentified as 18:0, 18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETAand EPA; and the composition of each is presented as a % of the totalfatty acids.

TABLE 46 Lipid Composition In Yarrowia Strain Y2067U Engineered ToOverexpress M. alpina DGAT1 Total Fatty Acids Strain 18:0 18:1 18:2 GLADGLA ARA ETA EPA Y2067U + pZUF-MOD-1 #1 1.31 6.92 12.03 23.11 5.72 1.053.80 13.20 Y2067U + pZUF-MOD-1 #2 1.39 6.83 12.15 21.99 5.83 1.07 3.8213.47 Y2067U + pMDGAT1-17 #1 0.89 7.13 10.87 24.88 5.82 1.19 3.97 14.09Y2067U + pMDGAT1-17 #2 0.86 7.20 10.25 22.42 6.35 1.26 4.38 15.07

As demonstrated above, expression of the M. alpina DGAT1 from plasmidpMDGAT1-17 increased the % EPA from ˜13.3% in the “control” strains to˜14.1% (“Y2067U+pMDGAT1-17#1”) and ˜15.1% (“Y2067U+pMDGAT1-17#2”),respectively. An additional increase in EPA would be expected, if thenative Yarrowia lipolytica DGAT1 (SEQ ID NOs:122 and 123) wereknocked-out in e.g., strain Y2067U+pMDGAT1-17.

Example 19 Mortierella alpina DGAT2 Increases Percent PUFAs

The present Example describes increased EPA biosynthesis andaccumulation in Yarrowia lipolytica strain Y2067U (Example 13) that wastransformed to co-express the M. alpina DGAT2 cDNA (SEQ ID NO:136). Itis contemplated that a Y. lipolytica host strain engineered to produceDHA via either the Δ6 desaturase/Δ6 elongase pathway or the Δ9elongase/Δ8 desaturase pathway could demonstrate increased DHAbiosynthesis and accumulation, if the M. alpina DGAT2 was similarlyco-expressed therein (e.g., in strain Y3000).

The M. alpina DGAT2 ORF was cloned into plasmid pZUF17 as follows.First, the ORF was PCR-amplified using primers MDGAT-F and MDGAT-R1 (SEQID NOs:407 and 408) from the M. alpina cDNA (supra, Example 16). Theexpected 1015 bp fragment was isolated, purified, digested with Nco Iand Not I and cloned into Nco I-Not I cut pZUF17 vector (SEQ ID NO:162;FIG. 9B), such that the gene was under the control of the Y. lipolyticaFBAIN promoter and the PEX20-3′ terminator region. Correct transformantswere confirmed by restriction analysis of miniprep DNA and the resultantplasmid was designated as “pMDGAT2-17” (SEQ ID NO:194).

Y. lipolytica strain Y2067U (from Example 13) was transformed withpMDGAT2-17 and pZUF-MOD-1 (supra, Example 17), respectively, accordingto the General Methods. Transformants were grown for 2 days in syntheticMM supplemented with amino acids, followed by 4 days in HGM. The fattyacid profile of two transformants containing pMDGAT2-17 and twotransformants containing pZUF-MOD-1 are shown below based on GC analysis(as described in the General Methods). Fatty acids are identified as18:0, 18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETA and EPA; and thecomposition of each is presented as a % of the total fatty acids.

TABLE 47 Lipid Composition In Yarrowia strain Y2067U Engineered ToOverexpress M. alpina DGAT2 Total Fatty Acids Strain 18:0 18:1 18:2 GLADGLA ARA ETA EPA Y2067U + pZUF-MOD-1 #1 1.31 6.92 12.03 23.11 5.72 1.053.80 13.20 Y2067U + pZUF-MOD-1 #2 1.39 6.83 12.15 21.99 5.83 1.07 3.8213.47 Y2067U + pMDGAT2-17 #1 0.00 7.47 10.77 25.30 5.70 1.43 3.45 15.12Y2067U + pMDGAT2-17 #2 1.45 7.79 9.96 25.16 6.06 1.25 3.99 15.37

Expression of the M. alpina DGAT2 from plasmid pMDGAT2-17 increased the% EPA from ˜13.3% in the “control” strains to ˜15.25%(“Y2067U+pMDGAT2-17”). An additional increase in EPA would be expected,if the native Yarrowia lipolytica DGAT2 (SEQ ID NOs:130-135) wereknocked-out in e.g., strain Y2067U+pMDGAT2-17.

Example 20 Mortierella alpina GPAT Increases Percent PUFAs

The present Example describes increased DGLA biosynthesis andaccumulation (and reduced quantities of 18:1) in Yarrowia lipolyticastrain Y2107U1 (Example 14) that was transformed to co-express the M.alpina GPAT ORF (SEQ ID NO:138). It is contemplated that a Y. lipolyticahost strain engineered to produce DHA via either the Δ6 desaturase/Δ6elongase pathway or the Δ9 elongase/Δ8 desaturase pathway coulddemonstrate increased DHA biosynthesis and accumulation, if the M.alpina GPAT was similarly co-expressed therein (e.g., in strain Y3000).

Identification of a M. aloina GPAT Using Degenerate PCR Primers

Based on sequences of GPAT from Aspergillus nidulans (GenBank AccessionNo. EAA62242) and Neurospora crassa (GenBank Accession No.XP_(—)325840), the following primers were designed for degenerate PCR:

MGPAT-N1 (SEQ ID NO: 409) CCNCAYGCNAAYCARTTYGT MGPAT-NR5 (SEQ ID NO:410) TTCCANGTNGCCATNTCRTC [Note: The nucleic acid degeneracy code usedfor SEQ ID NOs: 409 and 410 was as follows: R = A/G; Y = C/T; and N= A/C/T/G.]

PCR amplification was carried out in a Perkin Elmer GeneAmp 9600 PCRmachine using TaKaRa ExTaq premix Taq polymerase (TaKaRa Bio Inc., Otsu,Shiga, Japan). Amplification was carried out as follows: 30 cycles ofdenaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec andelongation at 72° C. for 90 sec, followed by a final elongation cycle at72° C. for 7 min.

A fragment of ˜1.2 kB was obtained (SEQ ID NO:140). This fragment waspurified with a Qiagen QiaQuick PCR purification kit, cloned into theTOPO® cloning vector pCR2.1-TOPO (Invitrogen), and sequenced. Theresultant sequence, when translated, had homology to known GPATs, basedon BLAST program analysis.

Based on the sequence of the 1212 bp cDNA fragment, the 5′ and 3′ endregions of the M. alpina GPAT were cloned by PCR amplification andgenome walking techniques. This enabled assembly of a contig,corresponding to the −1050 bp to +2885 bp region of the M. alpina GPAT(SEQ ID NO:141). This contig included the entire coding region of GPATand four introns (SEQ ID NOs:145, 146, 147 and 148).

Specifically, the M. alpina cDNA sample described in Example 16 (1 μl)was used as a template for amplification of the 3′-end of the GPAT.MGPAT-5N1 (SEQ ID NO:411) and CDSIII/3′ (SEQ ID NO:392) were used asprimers. PCR amplification was carried out in a Perkin Elmer GeneAmp9600 PCR machine using TaKaRa ExTaq premix Taq polymerase (TaKaRa BioInc., Otsu, Shiga, Japan). Amplification was carried out as follows: 30cycles of denaturation at 94° C. for 30 sec, annealing at 55° C. for 30sec and elongation at 72° C. for 120 sec, followed by a final elongationcycle at 72° C. for 7 min.

The PCR product was diluted 1:10, and 1 μl of diluted PCR product wasused as template for the second round of amplification, using MGPAT-5N2(SEQ ID NO:412) and CDSIII/3′ as primers. The conditions were exactlythe same as described above. The second round PCR product was againdiluted 1:10 and 1 μl of the diluted PCR product used as template for athird round of PCR, using MGPAT-5N3 (SEQ ID NO:413) and CDSIII/3′ asprimers. The PCR conditions were again the same.

A ˜1 kB fragment was generated in the third round of PCR. This fragmentwas purified with a Qiagen PCR purification kit and cloned intopCR2.1-TOPO vector for sequence analysis. Results from sequence analysisshowed that this 965 bp fragment (SEQ ID NO:142) corresponded with the3′-end of the GPAT gene.

A Clontech Universal GenomeWalker™ kit was used to obtain a piece ofgenomic DNA corresponding to the 5′-end region of the M. alpina GPAT.Briefly, 2.5 μg each of M. alpina genomic DNA was digested with DraI,EcoRV, PvuII or StuI individually, the digested DNA samples werepurified using Qiagen Qiaquick PCR purification kits and eluted with 30μl each of kit buffer EB, and the purified samples were then ligatedwith Genome Walker adaptor (SEQ ID NOs:414 [top strand] and 415 [bottomstrand]), as shown below:

5′-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT-3′                                    3′-H2N-CCCGACCA-5′Each ligation reaction mixture contained 1.9 μl of 25 μM Genome Walkeradaptor, 1.6 μl 10×ligation buffer, 0.5 μl T4 DNA ligase and 4 μl of oneof the purified digested genomic DNA samples. The reaction mixtures wereincubated at 16° C. overnight. The reaction was terminated by incubationat 70° C. for 5 min. Then, 72 μl of 10 mM TrisHCl, 1 mM EDTA, pH 7.4buffer was added to each ligation reaction mix.

Four separate PCR reactions were performed, each using one of the fourligation mixtures as template. The PCR reaction mixtures contained 1 μlof ligation mixture, 0.5 μl of 20 μM MGPAT-5-1A (SEQ ID NO:416), 1 μl of10 μM kit primer AP1 (SEQ ID NO:417), 22.5 μl water, and 25 μl ExTaqpremix Taq 2×PCR solution (TaKaRa). The PCR reactions were carried outfor 32 cycles using the following conditions: denaturation at 94° C. for30 sec, annealing at 55° C. for 30 sec, and elongation at 72° C. for 180sec. A final elongation cycle at 72° C. for 7 min was carried out,followed by reaction termination at 4° C.

The products of each PCR reaction were diluted 1:50 individually andused as templates for a second round of PCR. Each reaction mixturecontained 1 μl of one of the diluted PCR product as template, 0.5 μl of20 μM MGPAT-3N1 (SEQ ID NO:418), 21 μl of 10 μM kit primer AP2 (SEQ IDNO:419), 22.5 μl water and 25 μl of ExTaq premix Taq 2×PCR solution(TaKaRa). PCR reactions were carried out for 32 cycles using the samethermocycler conditions described above.

A DNA fragment was obtained from the second round of PCR. This fragmentwas purified and cloned into pCR2.1-TOPO and sequenced. Sequenceanalysis showed that the 1908 bp fragment (SEQ ID NO:143) was the 5′-endof the M. alpina GPAT gene.

Similarly, a 966 bp fragment (SEQ ID NO:144) was obtained by two roundsof genome walking as described above, except using primer MGPAT-5N1 asthe gene specific primer for the first round of PCR and primer MGPAT-5N2as the gene specific primer for the second round. This fragment was alsopurified, cloned into pCR2.1-TOPO and sequenced. Sequence analysisshowed that it contained a portion of the GPAT gene; however, thefragment was not long enough to extend to either end of the gene.Comparison with the 3′ cDNA sequence (SEQ ID NO:142) showed that thelast 171 bp of the ORF was not included.

Assembly of the Full-Length GPAT Sequence from Mortierella alpina

A 3935 bp sequence (SEQ ID NO:141) containing the complete GPAT gene(comprising a region extending 1050 bases upstream of the GPATtranslation initiation ‘ATG’ codon and extending 22 bases beyond theGPAT termination codon) was assembled from the sequences of the originalpartial cDNA fragment (SEQ ID NO:140), the 3′ cDNA fragment (SEQ IDNO:142), the internal genomic fragment (SEQ ID NO:144), and the 5′genomic fragment (SEQ ID NO:143) described above (graphicallyillustrated in FIG. 19). Included in this region is the 2151 bp GPATORF. The complete nucleotide sequence of the M. alpina GPAT ORF from‘ATG’ to the stop codon ‘TAG’ is provided as SEQ ID NO:138(corresponding to bases 1050 to 2863 of SEQ ID NO:141, excluding thefour introns (i.e., intron 1 [SEQ ID NO:145], corresponding to bases1195 to 1469 of SEQ ID NO:141; intron 2 [SEQ ID NO:146], correspondingto bases 1585 to 1839 of SEQ ID NO:141; intron 3 [SEQ ID NO:147],corresponding to bases 2795 to 2877 of SEQ ID NO:141 and intron 4 [SEQID NO:148], corresponding to bases 2940 to 3038 of SEQ ID NO:141). Thetranslated amino acid sequence (SEQ ID NO:139) showed homology with anumber of fungal, plant and animal GPATs.

More specifically, identity of the sequence was determined by conductingBLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., J.Mol. Biol. 215:403-410 (1993)) searches. The amino acid fragmentdescribed herein as SEQ ID NO:139 had 47% identity and 65% similaritywith the protein sequence of the putative GPAT of Ustilago maydis(GenBank Accession No. EAK84237), with an expectation value of 1e-152;additionally, SEQ ID NO:139 had 47% identity and 62% similarity with theGPAT of Aspergillus fumigatus (GenBank Accession No. EAL20089), with anexpectation value of 1e-142.

Construction of Plasmid pMGPAT-17, Comprising a FBAIN::MGPAT::PEX20-3′Chimeric Gene

The M. alpina GPAT ORF was cloned as follows. Primers MGPAT-cDNA-5 andMGPAT-cDNA-R (SEQ ID NOs:420 and 421) were used to amplify the GPAT ORFfrom the cDNA of M. alpina by PCR. The reaction mixture contained 1 μlof the cDNA, 1 μl each of the primers, 22 μl water and 25 μl ExTaqpremix 2×Taq PCR solution (TaKaRa Bio Inc., Otsu, Shiga, 520-2193,Japan). Amplification was carried out as follows: initial denaturationat 94° C. for 150 sec., followed by 30 cycles of denaturation at 94° C.for 30 sec, annealing at 55° C. for 30 sec and elongation at 72° C. for120 sec. A final elongation cycle at 72° C. for 10 min was carried out,followed by reaction termination at 4° C. An ˜2.2 kB DNA fragment wasobtained from the PCR reaction. It was purified using a Qiagen PCRpurification kit according to the manufacturer's protocol.

The purified PCR product was digested with BamHI and EcoRI, and a ˜470bp fragment was isolated by gel agarose electrophoresis and purifiedusing a Qiagen gel purification kit. Separately, the PCR product wasalso cut with EcoRI and NotI, and a 1.69 kB fragment isolated andpurified as above. The two fragments were ligated into BamHI and NotIcut pZUF-MOD-1 vector (SEQ ID NO:192; FIG. 18A), such that the gene wasunder the control of the Y. lipolytica FBAIN promoter and the PEX20-3′terminator region in the auto-replicating vector for expression in Y.lipolytica. Correct transformants were confirmed by restriction analysisof miniprep DNA and the resultant plasmid was designated as “pMGPAT-17”(SEQ ID NO:195; FIG. 18C).

Analysis of Lipid Composition in Transformant Y. lipolyticaOver-Expressing M. alpina GPAT

Y. lipolytica strain Y2107U1 (from Example 14) was transformed withplasmid pMGPAT-17 and plasmid pZUF-MOD-1 (supra, Example 17),respectively, according to the General Methods. Transformants were grownfor 2 days in synthetic MM supplemented with amino acids, followed by 4days in HGM. The fatty acid profile of two transformants containingpZUF-MOD-1 and four transformants containing pMGPAT-17, are shown belowin the Table, based on GC analysis (as described in the GeneralMethods). Fatty acids are identified as 18:0, 18:1 (oleic acid), 18:2(LA), GLA, DGLA, ARA, ETA and EPA; and the composition of each ispresented as a % of the total fatty acids.

TABLE 48 Lipid Composition In Yarrowia Strain Y2107U1 Engineered ToOver-Express M. alpina GPAT Total Fatty Acids Strain 18:0 18:1 18:2 GLADGLA ARA ETA EPA Y2107U1 + pZUF-MOD-1 #1 2.8 22.7 9.8 28.5 2.7 1.7 0.417.4 Y2107U1 + pZUF-MOD-1 #2 2.5 23.4 10.3 28.7 2.5 1.5 0.3 16.8Y2107U1 + pMGPAT-17 #1 3.2 14.8 11.7 29.8 5.6 2.0 0.3 18.4 Y2107U1 +pMGPAT-17 #2 2.9 16.3 11.7 28.3 6.1 1.8 0.4 16.9 Y2107U1 + pMGPAT-17 #32.1 14.3 10.8 27.5 7.2 1.4 0.4 17.4 Y2107U1 + pMGPAT-17 #4 2.7 15.7 11.529.1 6.3 1.7 0.4 17.3

As demonstrated above, expression of the M. alpina GPAT from pMGPAT-17increased the % DGLA from ˜2.5% in the “control” strains to 6.5%. Thelevel of 18:1 decreased from ˜23% to ˜16%. An additional increase inDGLA (or any other downstream PUFAs) would be expected, if the nativeYarrowia lipolytica GPAT was knocked-out in a transformant strainexpressing pMGPAT-17.

Example 21 Mortierella alpina Fatty Acid Elongase “ELO3” IncreasesPercent PUFAs

The present Example describes 35% more C18 fatty acids (18:0, 18:1, 18:2and GLA) and 31% less C16 fatty acids in Yarrowia lipolytica strainY2031 (Example 7) that was transformed to co-express the M. alpinaC_(16/18) fatty acid elongase (“ELO3”; SEQ ID NO:86), relative tocontrol strains. It is contemplated that ELO3 (which could optionally becodon-optimized for increased expression), could push carbon flux intoeither the engineered Δ6 desaturase/Δ6 elongase pathway or the Δ9elongase/Δ8 desaturase pathway as a means to increase production of thedesired PUFA, i.e., DHA. For example, a chimeric gene comprising thisC_(16/18) fatty acid elongase could readily be introduced into e.g.,strain Y3000.

Sequence Identification of a M. alpina C_(16/18) Fatty Acid Elongase

A cDNA fragment (SEQ ID NO:88) encoding a portion of a M. alpina fattyacid elongase was identified from among 9,984 M. alpina cDNA sequences(Example 16). This fragment bore significant homology to a number offatty acid elongases and thus was tentatively identified as an elongase.

The results of the BLAST comparison summarizing the sequence to whichSEQ ID NO:88 had the most similarity are reported according to the %identity, % similarity, and Expectation value. Specifically, thetranslated amino acid sequence of SEQ ID NO:88 had 32% identity and 46%similarity with the protein sequence of a potential fatty acid elongasefrom Candida albicans SC5314 (GenBank Accession No. EAL04510.1,annotated therein as one of three potential fatty acid elongase genessimilar to S. cerevisiae EUR4, FEN1 and ELO1), with an expectation valueof 4e-13. Additionally, SEQ ID NO:88 had 35% identity and 53% similaritywith ELO1 from Saccharomyces cerevisiae (GenBank Accession No.NC_(—)001142, bases 67849-68781 of chromosome X). The S. cerevisiae ELO1is described as a medium-chain acyl elongase, that catalyzescarboxy-terminal elongation of unsaturated C12-C16 fatty acyl-CoAs toC16-C18 fatty acids.

On the basis of the homologies reported above, the Yarrowia lipolyticagene product of SEQ ID NO:88 was designated herein as “elongase 3” or“ELO3”.

Analysis of the partial fatty acid elongase cDNA sequence (SEQ ID NO:88)indicated that the 5′ and 3′-ends were both incomplete. To obtain themissing 3′ region of the M. alpina ELO3, a Clontech UniversalGenomeWalker™ kit was used (as described in Example 20). Specifically,the same set of four ligation mixtures were used for a first round ofPCR, using the same components and conditions as described previously,with the exception that MA Elong 3′1 (SEQ ID NO:422) and AP1 were usedas primers (i.e., instead of primers MGPAT-5-1A and AP1). The secondround of PCR used MA Elong 3′2 (SEQ ID NO:423) and AP2 as primers. A1042 bp DNA fragment was obtained from the second round of PCR (SEQ IDNO:89). This fragment was purified and cloned into pCR2.1-TOPO andsequenced. Sequence analysis showed that the fragment contained the3′-end of ELO3, including ˜640 bp downstream of the ‘TAA’ stop codon ofthe gene.

The same set of four ligation mixtures used in the Clontech 3′-end RACE(supra) were also used to obtain the 5′-end region of the M. alpinaELO3. Specifically, a first round of PCR using the same components andconditions as described above was conducted, with the exception that MAElong 5′1 (SEQ ID NO:424, nested at the 5′ end) and AP1 were used asprimers (i.e., instead of primers MA Elong 3′1 and AP1). The secondround of PCR used MA Elong 5′2 (SEQ ID NO:425, nested at the 5′ end) andAP2 as primers. A 2223 bp DNA fragment (SEQ ID NO:90) was obtained. Itwas purified and cloned into pCR2.1-TOPO and sequenced. Analysis of thesequence showed that it contained the 5′-region of the ELO3 gene.

Thus, the entire cDNA sequence of the M. alpina ELO3 (SEQ ID NO:91) wasobtained by combining the original partial cDNA sequence (SEQ ID NO:88)with the overlapping 5′ and 3′ sequences obtained by genome walking (SEQID NOs:90 and 89, respectively; graphically illustrated in FIG. 20).This yielded a sequence of 3557 bp, identified herein as SEQ ID NO:91,comprising: 2091 bp upstream of the putative ‘ATG’ translationinitiation codon of ELO3; the 828 bp ELO3 ORF (i.e., SEQ ID NO:86,corresponding to bases 2092-2919 of SEQ ID NO:91); and, 638 bpdownstream of the ELO3 stop codon (corresponding to bases 2920-3557 ofSEQ ID NO:91).

The corresponding genomic sequence of the M. alpina ELO3 is longer thanthe cDNA fragment provided as SEQ ID NO:91. Specifically, a 542 bpintron (SEQ ID NO:92) was found in the genomic DNA containing the ELO3gene at 318 bp of the ORF; thus, the genomic DNA fragment, providedherein as SEQ ID NO:93, is 4,099 bp (FIG. 20).

The translated ELO3 protein sequence (SEQ ID NO:87) had the followinghomology, based on BLAST program analysis: 37% identity and 51%similarity to the potential fatty acid elongase from Candida albicansSC5314 (GenBank Accession No. EAL04510.1), with an expectation value of4e-43. Additionally, the translated ELO3 shared 33% identity and 44%similarity with the protein sequence of XP_(—)331368 (annotated thereinas a “hypothetical protein”) from Neurospora crassa, with an expectationvalue of 3e-44.

Construction of Plasmid pZUF6S-E3WT, Comprising a FBAIN::ELO3::PEX16-3′Chimeric Gene

The M. alpina fatty acid ELO3 ORF was cloned as follows. Primers MAElong 5′ NcoI 3 and MA Elong 3′ NotI (SEQ ID NOs:426 and 427) were usedto amplify the ELO3 ORF from the cDNA of M. alpina (Example 16) by PCR.The reaction mixture contained 1 μl of the cDNA, 1 μl each of theprimers, 22 μl water and 25 μl ExTaq premix 2×Taq PCR solution (TaKaRa).Amplification was carried out as follows: initial denaturation at 94° C.for 30 sec, followed by 32 cycles of denaturation at 94° C. for 30 sec,annealing at 55° C. for 30 sec and elongation at 72° C. for 120 sec. Afinal elongation cycle at 72° C. for 7 min was carried out, followed byreaction termination at 4° C. An ˜830 bp DNA fragment was obtained fromthe PCR reaction. It was purified using a Qiagen (Valencia, Calif.) PCRpurification kit according to the manufacturer's protocol. The purifiedPCR product was divided into two aliquots, wherein one was digested withNcoI and NspI, while the other with NspI and NotI. The ˜270 bp NcoI-NspIand ˜560 bp NspI-NotI fragments were cloned into Nco I-Not I cutpZF5T-PPC vector (FIG. 13B; SEQ ID NO:170) by three-piece ligation, suchthat the gene was under the control of the Y. lipolytica FBAIN promoterand the PEX16-3′ terminator region (GenBank Accession No. U75433) in theauto-replicating vector for expression in Y. lipolytica. Correcttransformants were confirmed by miniprep analysis and the resultantplasmid was designated as “pZF5T-PPC-E3” (SEQ ID NO:196).

Plasmid pZF5T-PPC-E3 was digested with ClaI and PacI and the ˜2.2 kBband (i.e., the FBAIN::ELO 3::PEX16-3′ fragment) was purified from anagarose gel using a Qiagen gel extraction kit. The fragment was clonedinto ClaI-PacI cut pZUF6S (FIG. 21A; SEQ ID NO:197), an auto-replicationplasmid containing the Mortierella alpina Δ6 desaturase ORF (“D6S”;GenBank Accession No. AF465281) under the control of the FBAIN promoterwith a Pex20-3′ terminator (i.e., a FBAIN::D6S::Pex20 chimeric gene) anda Ura3 gene. Correct transformants were confirmed by miniprep analysisand the resultant plasmid was designated as “pZUF6S-E3WT” (FIG. 21B; SEQID NO:198).

Analysis of Lipid Composition in Transformant Y. lipolyticaOver-Expressing the M. alpina ELO3

Y. lipolytica strain Y2031 (Example 7) was transformed with plasmidpZUF6S (control, comprising a FBAIN::D6S::Pex20 chimeric gene) andplasmid pZUF6S-E3WT (comprising a FBAIN::D6S::Pex20 chimeric gene andthe FBAIN::ELO 3::PEX16 chimeric gene) according to the General Methods.Transformants were grown for 2 days in synthetic MM supplemented withamino acids, followed by 4 days in HGM. The fatty acid profile of sixclones containing pZUF6S (clones #1-6, from a single transformation) and22 clones (from four different transformations [i.e., #3, 5, 6, and 7])containing pZUF6S-E3WT are shown below in Table 49, based on GC analysis(as described in the General Methods). Fatty acids are identified as16:0 (palmitate), 16:1 (palmitoleic acid), 18:0, 18:1 (oleic acid), 18:2(LA) and GLA; and the composition of each is presented as a % of thetotal fatty acids.

TABLE 49 Lipid Composition In Yarrowia Strain Y2031 Engineered ToOver-Express M. alpina ELO3 Y. lipolytica Strain Y2031 TransformantFatty Acid Composition (% Of Total Fatty Acids) And/Or Clone No. 16:016:1 18:0 18:1 18:2 GLA pZUF6S #1 (control) 9.0 23.2 1.2 38.2 19.8  6.9pZUF6S #2 (control) 10.1  23.4 1.4 39.0 17.5  7.1 pZUF6S #3 (control)9.7 22.7 1.4 39.0 20.2  7.0 pZUF6S #4 (control) 8.5 24.1 0.0 40.8 19.8 6.9 pZUF6S #5 (control) 9.8 22.4 1.7 39.1 20.2  6.8 pZUF6S #6 (control)9.1 22.7 1.9 39.9 19.7  6.6 pZUF6S-E3WT #3-1 8.9 17.3 4.1 36.5 21.6 11.6pZUF6S-E3WT #3-2 8.8 17.8 3.7 36.9 21.3 11.5

pZUF6S-E3WT #3-6 8.5 19.9 4.4 37.8 17.1 12.3 pZUF6S-E3WT #5-1 8.6 17.64.0 37.6 21.1 11.1 pZUF6S-E3WT #5-2 8.8 17.1 3.9 37.6 21.3 11.2pZUF6S-E3WT #5-3 9.1 17.1 3.5 37.6 21.5 11.1 pZUF6S-E3WT #5-4 8.8 17.94.3 38.0 19.3 11.7 pZUF6S-E3WT #5-5 9.2 16.1 4.4 37.0 21.6 11.7

pZUF6S-E3WT #6-1 9.4 16.9 4.6 36.6 21.5 11.0 pZUF6S-E3WT #6-2 9.8 16.24.1 36.5 21.9 11.6 pZUF6S-E3WT #6-3 9.4 17.0 4.4 36.2 21.8 11.3pZUF6S-E3WT #6-4 8.3 16.6 4.2 36.9 21.9 12.2 pZUF6S-E3WT #6-5 8.8 18.55.5 36.0 17.8 13.4 pZUF6S-E3WT #6-6 8.7 19.5 5.2 35.4 18.1 13.2

pZUF6S-E3WT #7-2 8.0 17.7 4.0 37.7 20.9 11.7

pZUF6S-E3WT #7-5 8.3 17.0 4.7 36.7 21.2 12.1 pZUF6S-E3WT #7-6 8.0 18.04.8 36.3 20.8 12.1

Some of the samples (labeled in bold and italics) deviated from expectedreadings. Specifically, neither Y2031+pZUF6S-E3WT #3-3 norY2031+pZUF6S-E3WT #5-6 produced GLA. Similarly, Y2031+pZUF6S-E3WT #7-1,#7-3 and #7-4 had GC errors, wherein the 16:0 and 16:1 peaks were readby the GC as a single peak. As a result of these variant results, Table50 reports the average lipid in the control and transformant strainsexpressing ELO3. Specifically, Table 50 shows the averages from thefatty acid profiles in Table 49, although the lines indicated by boldand italics as being incorrect in Table 49 were not included whencalculating these averages. “Total C16” represents the sum of theaverage areas of 16:0 and 16:1, while “Total C18” reflects the sum ofthe average areas of 18:0, 18:1, 18:2 and GLA.

TABLE 50 Average Lipid Composition In Yarrowia Strain Y2031 EngineeredTo Over-Express M. alpina ELO3 Y. lipolytica Average Fatty AcidComposition Strain Y2031 (% Of Total Fatty Acids) Total TotalTransformant 16:0 16:1 18:0 18:1 18:2 GLA C16 C18 pZUF6S 9.4 23.1 1.339.3 19.5 6.9 32.4 67.1 (control) pZUF6S- 8.7 18.3 4.1 37.1 20.0 11.827.0 73.0 E3WT #3 pZUF6S- 8.9 17.2 4.0 37.6 21.0 11.4 26.1 73.9 E3WT #5pZUF6S- 9.1 17.5 4.6 36.3 20.5 12.1 26.5 73.5 E3WT #6 pZUF6S- 8.1 17.64.5 36.9 21.0 12.0 25.6 74.4 E3WT #7

Based on the data reported above, overexpression of the M. alpina ELO3resulted in an increased percentage of C18 and a reduced percentage ofC16 when co-expressed with a M. alpina Δ6 desaturase in Yarrowialipolytica strain Y2031, relative to a control strain of Y2031overexpressing the M. alpina Δ6 desaturase only. This indicated that theM. alpina ELO3 was indeed a C_(16/18) fatty acid elongase.

Example 22 Yarrowia C_(16/18) Fatty Acid Elongase “YE2” IncreasesPercent PUFAs

The present Example describes increased GLA biosynthesis andaccumulation in Yarrowia lipolytica strain Y2031 (Example 7) that wastransformed to co-express the Y. lipolytica C_(16/18) fatty acidelongase (“YE2”; SEQ ID NO:94). It is contemplated that the YE2 elongasecould push carbon flux into either the engineered Δ6 desaturase/Δ6elongase pathway or the Δ9 elongase/Δ8 desaturase pathway as a means toincrease production of the desired PUFA, i.e., DHA. For example, achimeric gene comprising this C_(16/18) fatty acid elongase couldreadily be introduced into e.g., strain Y3000.

Sequence Identification of a Yarrowia lipolytica C_(16/18) Fatty AcidElongase

A novel fatty acid elongase candidate from Y. lipolytica was identifiedby sequence comparison using the rat Elo2 C_(16/18) fatty acid elongaseprotein sequence (GenBank Accession No. AB071986; SEQ ID NO:84) as aquery sequence. Specifically, this rElo2 query sequence was used tosearch GenBank and the public Y. lipolytica protein database of the“Yeast project Genolevures” (Center for Bioinformatics, LaBRI, TalenceCedex, France) (see also Dujon, B. et al., Nature 430 (6995):35-44(2004)). This resulted in the identification of a homologous sequence,GenBank Accession No. CAG77901 (SEQ ID NOs:94 and 95), annotated as an“unnamed protein product”). This gene was designated as YE2.

Comparison of the Yarrowia YE2 amino acid sequences to public databases,using a BLAST algorithm (Altschul, S. F., et al., Nucleic Acids Res.25:3389-3402 (1997)), revealed that the most similar known amino acidsequence was that from Candida albicans SC5314 (SEQ ID NO:96, GenBankAccession No. EAL04510), annotated as a probable fatty acid elongase.The proteins shared about 40% identity and scored at 236 with an E valueof 7e-61.

Isolation of Yarrowia YE2 Gene

The coding region of the YE2 gene was amplified by PCR using Yarrowiagenomic DNA as template and oligonucleotides YL597 and YL598 (SEQ IDNOs:428 and 429) as primers. The PCR reaction was carried out in a 50 μltotal volume, as described in the General Methods. The thermocyclerconditions were set for 35 cycles at 95° C. for 1 min, 56° C. for 30sec, 72° C. for 1 min, followed by a final extension at 72° C. for 10min. The PCR products of the YE2 coding region were purified, digestedwith NcoI/NotI, and then ligated with NcoI/NotI digested pZKUGPYE1-N(infra, Example 23; see also FIG. 21C, SEQ ID NO:199) to generatepZKUGPYE2 (FIG. 21D, SEQ ID NO:200). The addition of a NcoI site aroundthe ‘ATG’ translation initiation codon changed the second amino acid ofYE2 from L to V.

The ClaI/NotI fragment of pZKUGPYE2 (containing the GPAT promoter andYE2 coding region) and a NotI/PacI fragment containing the Acoterminator (prepared by PCR amplifying the ACO 3′ terminator withprimers YL325 and YL326 [SEQ ID NOs:430 and 431] and then digesting withNotI/PacI), were directionally ligated with ClaI/PacI digested vectorpZUF6S to produce pZUF6YE2. The ClaI/NcoI fragment of pZKUT16(containing the TEF promoter) and the NcoI/PacI fragment of pZUF6YE2(containing the coding region of YE2 and the Aco terminator) weresubsequently directionally ligated with ClaI/PacI digested vector pZUF6Sto produce pZUF6TYE2 (SEQ ID NO:201).

Analysis of Lipid Composition in Transformant Y. lipolyticaOver-Expressing YE2

Plasmid pZUF6S (FIG. 21A, SEQ ID NO:197) and pZUF6TYE2 (SEQ ID NO:201)were used to separately transform Yarrowia strain Y2031. The componentsof these two plasmids are described in Table 51 and 52.

TABLE 51 Description of Plasmid pZUF6S (SEQ ID NO: 197) RE Sites AndNucleotides Within Description Of Fragment And Chimeric Gene SEQ ID NO:197 Components EcoRI/ClaI Yarrowia autonomous replicating sequence 18(3114-4510) (ARS18; GenBank Accession No. M91600) SalI/PacI YarrowiaUra3 gene (GenBank Accession No. (6022-4530) AJ306421) EcoRI/BsiWIFBAIN::Δ6S::Pex20, comprising: (6063-318) FBAIN: FBAIN promoter (SEQ IDNO: 214) Δ6S: codon-optimized Δ6 desaturase gene (SEQ ID NO: 3), derivedfrom Mortierella alpina (GenBank Accession No. AF465281) Pex20: Pex20terminator sequence from Yarrowia Pex20 gene (GenBank Accession No.AF054613)

TABLE 52 Description of Plasmid pZUF6TYE2 (SEQ ID NO: 201) RE Sites AndNucleotides Within Description Of Fragment And Chimeric Gene SEQ ID NO:201 Components EcoRI/ClaI Yarrowia autonomous replicating sequence 18(7461-8857) (ARS18; GenBank Accession No. M91600) SalI/PacI YarrowiaUra3 gene (GenBank Accession No. (1907-415) AJ306421) EcoRI/BsiWIFBAIN::Δ6S::Pex20: as described for pZUF6 (1948-4665) (supra) ClaI/PacITEF::YE2::Aco, comprising: (8857-415) TEF: TEF promoter (GenBankAccession No. AF054508) YE2: coding region of Yarrowia YE2 gene (SEQ IDNO: 94; GenBank Accession No. CAG77901) Aco: Terminator sequence ofYarrowia Aco3 gene (GenBank Accession No. AJ001301)

Y. lipolytica strain Y2031 (Example 7) was transformed with plasmidpZUF6S (control) and plasmid pZUF6TYE2 according to the General Methods.Transformants were grown for 2 days in liquid MM. The fatty acid profileof eight colonies each containing pZUF6S or pZUF6YE2 are shown below inTable 53, based on GC analysis (as described in the General Methods).Fatty acids are identified as 16:0 (palmitate), 16:1 (palmitoleic acid),18:0, 18:1 (oleic acid), 18:2 (LA) and GLA; and the composition of eachis presented as a % of the total fatty acids.

TABLE 53 Comparison Of Fatty Acid Composition In Yarrowia Strain Y2031Transformed With pZUF6S And pZUF6TYE2 Y. lipolytica Strain Y2031 FattyAcid Composition (% Of Total Fatty Acids) Transformants 16:0 16:1 18:018:1 18:2 GLA pZUF6S #1 (control) 15.4 13.8 2.5 34.1 16.8 8.3 pZUF6S #2(control) 15.2 12.8 3.0 36.5 16.4 8.3 pZUF6S #3 (control) 15.1 12.2 3.236.5 17.1 8.5 pZUF6S #4 (control) 15.2 12.8 3.1 36.3 16.6 8.4 pZUF6S #5(control) 14.9 10.9 3.6 37.4 18.0 8.7 pZUF6S #6 (control) 14.8 10.1 4.237.6 18.7 8.6 pZUF6S #7 (control) 14.7 11.9 3.0 36.0 17.8 9.1 pZUF6S #8(control) 14.9 12.6 2.9 35.9 17.3 8.8 Average 15.0 12.1 3.2 36.3 17.38.6 pZUF6TYE2 #1 13.1 8.4 4.4 42.4 16.8 9.7 pZUF6TYE2 #2 13.1 7.6 5.340.8 18.6 9.8 pZUF6TYE2 #3 13.5 8.1 4.6 39.2 19.0 10.6 pZUF6TYE2 #4 13.47.4 5.7 39.9 18.7 9.8 pZUF6TYE2 #5 13.4 8.4 5.5 45.2 14.3 7.6 pZUF6TYE2#6 13.4 7.4 5.5 39.3 19.2 10.5 pZUF6TYE2 #7 13.4 8.6 4.4 40.6 17.9 9.9pZUF6TYE2 #8 13.2 7.5 5.4 41.2 18.0 9.7 Average 13.3 8.0 5.0 41.1 17.89.7

GC analyses showed that there were about 27.1% C16 (C16:0 and C16:1) and62.2% C18 (C18:0, C18:1, C18:2 and GLA) of total lipids produced in theY2031 transformants with pZUF6S; there were about 21.3% C16 and 73.6%C18 produced in the Y2031 transformants with pZUF6TYE2. Thus, the totalamount of C16 was reduced about 21.4%, and the total amount of C18 wasincreased about 18% in the pZUF6TYE2 transformants (as compared with thetransformants with pZUF6S). These data demonstrated that YE2 functionsas a C_(16/18) fatty acid elongase to produce C18 fatty acids inYarrowia. Additionally, there was about 12.8% more GLA produced in thepZUF6TYE2 transformants relative to the GLA produced in pZUF6Stransformants. These data suggested that the YE2 elongase could pushcarbon flux into the engineered PUFA pathway to produce more finalproduct (i.e., GLA).

Example 23 Yarrowia C_(14/16) Fatty Acid Elongase “YE1” IncreasesPercent PUFAs

The present Example describes increased GLA biosynthesis andaccumulation in Y. lipolytica strain Y2031 (Example 7) that wastransformed to co-express the Y. lipolytica C_(14/16) fatty acidelongase (“YE1”; SEQ ID NO:97). It is contemplated that the YE1 elongasecould push carbon flux into either the engineered Δ6 desaturase/Δ6elongase pathway or the Δ9 elongase/Δ8 desaturase pathway as a means toincrease production of the desired PUFA, i.e., DHA. Specifically, achimeric gene comprising this C_(14/16) fatty acid elongase couldreadily be introduced into e.g., strain Y3000.

Sequence Identification of a Yarrowia lipolytica C_(14/16) Fatty AcidElongase

A novel fatty acid elongase candidate from Yarrowia lipolytica wasidentified by sequence comparison using the rat Elo2 C_(16/18) fattyacid elongase protein sequence (GenBank Accession No. ABO71986; SEQ IDNO:84) as a query sequence, in a manner similar to that used in Example22. This resulted in the identification of a homologous sequence,GenBank Accession No. CAG83378 (SEQ ID NOs:97 and 98), annotated as an“unnamed protein product”. This gene was designated as “YE1”.

Comparison of the Yarrowia YE1 amino acid sequences to public databases,using a BLAST algorithm (Altschul, S. F., et al., Nucleic Acids Res.25:3389-3402 (1997)), revealed that the most similar known sequence wasFEN1 from Neurospora crassa (GenBank Accession No. CAD70918; SEQ IDNO:99), a probable fatty acid elongase sharing about 60% identity toYE1.

Isolation of Yarrowia YE1 Gene

The DNA sequence of YE1 gene (SEQ ID NO:97) possesses an internal NcoIsite. In order to incorporate the Yarrowia translation motif around the‘ATG’ translation initiation codon of the YE1 gene, a two-step strategywas employed to PCR the entire YE1 gene from Yarrowia. Specifically,using Yarrowia genomic DNA as template, the first half of YE1 wasamplified by PCR using oligonucleotides YL567 and YL568 (SEQ ID NOs:432and 433) as primers, while the second half of the YE1 gene was amplifiedsimilarly using oligonucleotides YL569 and YL570 (SEQ ID NOs:434 and435) as primers. The PCR reactions were carried out in a 50 μl totalvolume, as described in the General Methods. The thermocycler conditionswere set for 35 cycles at 95° C. for 1 min, 56° C. for 30 sec, 72° C.for 1 min, followed by a final extension at 72° C. for 10 min. The PCRproducts corresponding to the 5′ portion of YE1 were purified and thendigested with NcoI and SacI to yield the YE1-1 fragment, while the PCRproducts of the 3′ portion of YE1 were purified and digested with SacIand NotI to yield the YE1-2 fragment. The YE1-1 and YE1-2 fragments weredirectly ligated with NcoI/NotI digested pZKUGPE1S (supra, Example 14)to generate pZKUGPYE1 (FIG. 22A, SEQ ID NO:202). The internal NcoI siteof YE1 was then mutated by site-directed mutagenesis using pZKUGPYE1 astemplate and oligonucleotides YL571 and YL572 (SEQ ID NOs:436 and 437)as primers to generate pZKUGPYE1-N (SEQ ID NO:199). Sequence analysisshowed that the mutation did not change the amino acid sequence of YE1.The addition of the NcoI site around the ATG translation initiationcodon changed the second amino acid of YE1 from S to A.

The ClaI/NcoI fragment of pZF5T-PPC (containing the FBAIN promoter) andthe NcoI/PacI fragment of pZKUGPYE1-N (containing the coding region ofYE1 and the Aco terminator) were directionally ligated withClaI/PacI-digested vector pZUF6S to produce pZUF6FYE1 (SEQ ID NO:203).

Analysis of Lipid Composition in Transformant Y. lipolyticaOver-Expressing YE1

Plasmids pZUF6S and pZUF6FYE1 (SEQ ID NO:203) were used to separatelytransform Yarrowia strain Y2031 (from Example 7) according to theGeneral Methods. The components of control plasmid pZUF6S (FIG. 21A; SEQID NO:197; comprising a FBAIN::D6S::Pex20 chimeric gene) were describedin Example 22. The components of pZUF6FYE1 (FIG. 22B; SEQ ID NO:203,comprising a FBAIN::D6S::Pex20 chimeric gene and the FBAIN::YE1::Acochimeric gene) are described in Table 54 below.

TABLE 54 Description Of Plasmid pZUF6FYE1 (SEQ ID NO: 203) RE Sites AndNucleotides Within Description Of Fragment And Chimeric Gene SEQ ID NO:203 Components EcoRI/ClaI Yarrowia autonomous replicating sequence 18(7047-8445) (ARS18; GenBank Accession No. M91600) SalI/PacI YarrowiaUra3 gene (GenBank Accession No. (1493-1) AJ306421) EcoRI/BsiWIFBAIN::Δ6S::Pex20: as described for pZUF6 (1534-4251) (supra, Example22) ClaI/PacI FBAIN::YE1::Aco, comprising: (8443-1) FBAIN: FBAINpromoter (SEQ ID NO: 214) YE1: coding region of Yarrowia YE1 gene (SEQID NO: 97; GenBank Accession No. CAG83378) Aco: Aco3 terminator sequencefrom Yarrowia Aco3 gene (Genbank Accession No. AJ001301)

Following transformation, transformants were grown for 2 days insynthetic MM supplemented with amino acids, followed by 4 days in HGM.The fatty acid profile of six clones containing pZUF6S and five clonescontaining pZUF6FYE1 are shown below in Table 55, based on GC analysis(as described in the General Methods). Fatty acids are identified as16:0 (palmitate), 16:1 (palmitoleic acid), 18:0, 18:1 (oleic acid), 18:2(LA) and GLA; and the composition of each is presented as a % of thetotal fatty acids.

TABLE 55 Comparison Of Fatty Acid Composition In Yarrowia Strain Y2031Transformed With pZUF6S And pZUF6FYE1 Fatty Acid Composition (% Of TotalFatty Acids) Transformants 16:0 16:1 18:1 18:2 GLA pZUF6S #1 (control)12.9 18.2 29.6 23.5 10.7 pZUF6S #2 (control) 12.6 18.6 29.6 23.8 10.3pZUF6S #3 (control) 13.0 17.8 29.8 23.9 10.6 pZUF6S #4 (control) 13.118.9 30.1 22.3 10.3 pZUF6S #5 (control) 13.0 17.8 29.6 23.4 10.9 pZUF6S#6 (control) 12.0 18.7 30.4 23.2 10.4 Average 12.8 18.3 29.9 23.4 10.5pZUF6FYE1 #1 17.4 21.9 20.4 19.2 16.9 pZUF6FYE1 #2 16.7 22.8 21.1 19.116.1 pZUF6FYE1 #3 19.8 20.7 22.8 17.0 15.8 pZUF6FYE1 #4 16.8 22.4 23.716.1 16.8 pZUF6FYE1 #5 17.7 21.6 21.2 18.0 17.2 Average 17.7 21.9 21.917.9 16.5

GC analyses measured about 31.1% C16 (C16:0+C16:1) of total lipidsproduced in the Y2031 transformants with pZUF6S, while there was about39.6% C16 produced in the Y2031 transformants with pZUF6FYE1. The totalamount of C16 increased about 26.7% in the pZUF6FYE1 transformants, ascompared to transformants with pZUF6S. Thus, these data demonstratedthat YE1 functions as a C_(14/16) fatty acid elongase to produce C16fatty acids in Yarrowia. Additionally, there was 57% more GLA producedin the pZUF6FYE1 transformants than in pZUF6S transformants, suggestingthat the YE1 elongase could push carbon flux into the engineered pathwayto produce more final product (i.e., GLA).

Example 24 Yarrowia lipolytica CPT1 Overexpression Increases PercentPUFAs

The present Example describes increased EPA biosynthesis andaccumulation in Yarrowia lipolytica strain Y2067U (Example 13) that wastransformed to overexpress the Y. lipolytica CPT1 cDNA (SEQ ID NO:150).PUFAs leading to the synthesis of EPA were also increased. It iscontemplated that a Y. lipolytica host strain engineered to produce DHAvia either the Δ6 desaturase/Δ6 elongase pathway or the Δ9 elongase/Δ8desaturase pathway could demonstrate increased DHA biosynthesis andaccumulation, if the Y. lipolytica CPT1 was similarly co-expressed(e.g., in strain 3000).

Y. lipolytica strain ATCC #20326 cDNA was prepared using the followingprocedure. Cells were grown in 200 mL YPD medium (2% Bacto-yeastextract, 3% Bactor-peptone, 2% glucose) for 1 day at 30° C. and thenpelleted by centrifugation at 3750 rpm in a Beckman GH3.8 rotor for 10min and washed twice with HGM. Washed cells were resuspended in 200 mLof HGM and allowed to grow for an additional 4 hrs at 30° C. Cells werethen harvested by centrifugation at 3750 rpm for 10 min in 4×50 mLtubes.

Total RNA was isolated using the Qiagen RNeasy total RNA Midi kit. Todisrupt the cells, harvested cells were resuspended in 4×600 μl of kitbuffer RLT (supplemented with β-mercaptoethanol, as specified by themanufacturer) and mixed with an equal volume of 0.5 mm glass beads infour 2 mL screwcap tubes. A Biospec Mini-beadbeater was used to breakthe cells for 2 min at the Homogenization setting. An additional 4×600μl buffer RLT was added. Glass beads and cell debris were removed bycentrifugation, and the supernatant was used to isolate total RNAaccording to manufacturer's protocol.

PolyA(+)RNA was isolated from the above total RNA sample using a QiagenOligotex mRNA purification kit according to the manufacturer's protocol.Isolated polyA(+)RNA was purified one additional round with the same kitto ensure the purity of mRNA sample. The final purified poly(A)+RNA hada concentration of 30.4 ng/μl.

cDNA was generated, using the LD-PCR method specified by BD-Clontech and0.1 μg of polyA(+)RNA sample, as described in Example 16, with theexception that the PCR thermocycler conditions used for 1^(st) strandcDNA synthesis were set for 95° C. for 20 sec, followed by 20 cycles of95° C. for 5 sec and 68° C. for 6 min. The PCR product was quantitatedby agarose gel electrophoresis and ethidium bromide staining.

The Y. lipolytica CPT1 cDNA was cloned as follows. Primers CPT1-5′-NcoIand CPT1-3′-NotI (SEQ ID NOs:438 and 439) were used to amplify the Y.lipolytica ORF from the cDNA of Y. lipolytica by PCR. The reactionmixture contained 0.5 μl of the cDNA, 0.5 μl each of the primers, 11 μlwater and 12.5 μl ExTaq premix 2×Taq PCR solution (TaKaRa Bio Inc.,Otsu, Shiga, 520-2193, Japan). Amplification was carried out as follows:initial denaturation at 94° C. for 300 sec, followed by 30 cycles ofdenaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec, andelongation at 72° C. for 60 sec. A final elongation cycle at 72° C. for10 min was carried out, followed by reaction termination at 4° C. A˜1190 bp DNA fragment was obtained from the PCR reaction. It waspurified using Qiagen's PCR purification kit according to themanufacturer's protocol. The purified PCR product was digested with NcoIand NotI, and cloned into Nco I-Not I cut pZUF17 vector (SEQ ID NO:162;FIG. 9B), such that the gene was under the control of the Y. lipolyticaFBAIN promoter and the PEX20-3′ terminator region. Correct transformantswere confirmed by miniprep analysis and the resultant plasmid wasdesignated as “pYCPT1-17” (SEQ ID NO:204).

To integrate the chimeric FBAIN::CPT1::PEX20 gene into the genome ofYarrowia lipolytica, plasmid pYCPT1-ZP2l7 was created by digestingpYCPT1-17 with NcoI and NotI, and isolating the ˜1190 bp fragment thatcontained the CPT1 ORF. This fragment was then cloned into pZP2l7+Ura(SEQ ID NO:205) digested with NcoI and NotI. As shown in FIG. 22C,plasmid pZP2l7+Ura is a Y. lipolytica integration plasmid comprising achimeric TEF::synthetic Δ17 desaturase (codon-optimized for Y.lipolytica)::Pex20-3′ gene and a Ura3 gene, for use as a selectablemarker. Correct transformants were confirmed by miniprep analysis andthe resultant plasmid was designated as “pYCPT1-ZP2l7” (SEQ ID NO:206).

Y. lipolytica strain Y2067U (from Example 13) was transformed withBssHII/BbuI digested pYCPT1-ZP2l7 and pZUF-MOD-1 (supra, Example 17),respectively, according to the General Methods. Transformants were grownfor 2 days in synthetic MM supplemented with amino acids, followed by 4days in HGM. The fatty acid profile of two transformants containingpZUF-MOD-1 and four transformants having pYCPT1-ZP2I7 integrated intothe genome are shown below in the Table, based on GC analysis (asdescribed in the General Methods). Fatty acids are identified as 18:0,18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETA and EPA; and thecomposition of each is presented as a % of the total fatty acids.

TABLE 56 Lipid Composition In Yarrowia Strain Y2067U Engineered ToOverexpress Y. lipolytica CPT1 Total Fatty Acids Strain 18:0 18:1 18:2GLA DGLA ARA ETA EPA Y2067U + pZUF-MOD-1 #1 1.3 6.9 12.0 23.1 5.7 1.13.8 13.2 Y2067U + pZUF-MOD-1 #2 1.4 6.8 12.1 22.0 5.8 1.1 3.8 13.5Y2067U + pYCPT1-ZP2I7 #1 0.6 8.0 8.2 27.4 7.1 1.6 4.1 15.7 Y2067U +pYCPT1-ZP2I7 #2 0.6 8.1 8.2 27.2 7.0 1.6 4.0 15.7 Y2067U + pYCPT1-ZP2I7#3 1.0 7.9 8.0 24.7 6.1 1.6 3.2 15.5 Y2067U + pYCPT1-ZP2I7 #4 0.6 7.18.6 25.5 6.9 1.8 4.0 16.0

As shown above, expression of the Y. lipolytica CPT1 under the controlof the strong FBAIN promoter, by genome integration, increased the % EPAfrom 13.4% in the “control” strains to 15.7-16%. Furthermore, GLA, DGLAand ARA levels also were increased.

Example 25 Sacchromyces cerevisiae ISC1 Increases Percent PUFAs

The present Example describes increased EPA biosynthesis andaccumulation in Yarrowia lipolytica strain M4 (Example 6) that wastransformed to co-express the S. cerevisiae ISC1 gene (SEQ ID NO:152).It is contemplated that a Y. lipolytica host strain engineered toproduce DHA via either the Δ6 desaturase/Δ6 elongase pathway or the Δ9elongase/Δ8 desaturase pathway could demonstrate increased DHAbiosynthesis and accumulation, if the S. cerevisiae ISC1 was similarlyco-expressed (e.g., in strain Y3000).

The S. cerevisiae ISC1 ORF was cloned into plasmid pZP2l7+Ura asfollows. First, the ORF was PCR-amplified using genomic DNA from S.cerevisiae strain S288C (Promega, Madison, Wis.) and primer pair Isc1Fand Isc1R (SEQ ID NOs:440 and 441). Primer Isc1F modified the wildtype5′ sequence of ISC1 from ‘ATGTACAA’ to ‘ATGGACAA’ in the amplified ORF,as it was necessary to incorporate a NcoI site and thereby keep ISC1 inframe. Amplification was carried out as follows: initial denaturation at94° C. for 120 sec, followed by 35 cycles of denaturation at 94° C. for30 sec, annealing at 50° C. for 30 sec and elongation at 68° C. for 120sec. A final elongation cycle at 68° C. for 10 min was carried out,followed by reaction termination at 4° C. A 1455 bp DNA fragment wasobtained from the PCR reaction for ISC1 and the PCR product size wasconfirmed by electrophoresis, using a 1% agarose gel (120 V for 30 min)and a 1 kB DNA standard ladder from Invitrogen (Carlsbad, Calif.).

The DNA was purified using a DNA Clean & Concentrator-5 kit from ZymoResearch Corporation (Orange, Calif.), per the manufacturer'sinstructions, and then digested with NcoI/NotI. The ISC1 fragment wasthen individually cloned into pZP2l7+Ura (SEQ ID NO:205; FIG. 22C)digested with NcoI and NotI. Correct transformants were confirmed by gelelectrophoresis and the resultant plasmid was designated as “pTEF::ISC1”(SEQ ID NO:207). Thus, this plasmid contained a DNA cassette comprisingthe following: 3′-POX2, URA3, TEF::ISC1::Pex20 and a POX2 promoterregion.

“Control” vector was prepared as follows. First, the S. cerevisiae pcl1ORF (encoding a protein involved in entry into the mitotic cell cycleand regulation of morphogenesis) was PCR amplified using genomic DNAfrom S. cerevisiae strain S288C and primer pair Pcl1F and Pcl1R (SEQ IDNOs:442 and 443). Amplification was carried out as described above. A861 bp DNA fragment was obtained from the PCR reaction for pcl1(confirmed by electrophoresis, supra). The DNA was purified using a DNAClean & Concentrator-5 kit and then digested with NcoI/NotI. Thefragment was then cloned into similarly digested pZP2I7+Ura. Correcttransformants were confirmed by gel electrophoresis and the resultantplasmid was designated as “pTEF::pcl1”. Plasmid pTEF::plc1 was thendigested with HincII to remove the pcl1 ORF. The remaining plasmid wasreligated, such that a linear DNA cassette comprising 3′-POX2, URA3,TEF::Pex20 and a POX2 promoter region resulted upon digestion withAscI/SphI.

Competent Y. lipolytica strain M4 cells (from Example 6) weretransformed with Asc1/Sph1-digested pTEF::ISC1 and “control”,respectively (wherein 5 μg of each plasmid had been subject todigestion). Transformation was accomplished using the Frozen EZ YeastTransformation II kit (Zymo Research) and transformants were selected onplates comprising YNB without Amino Acids (6.7 g/L; Becton, Dickinsonand Co., Sparks, Md. [Catalog #291940]), glucose (20 g/L) and agar (20g/L). Several hundred transformant colonies were obtained. Integrationof each DNA cassette into the Yarrowia lipolytica POX2 locus wasconfirmed by PCR using the genomic DNA from 5 independent transformantsfor ISC1.

Transformants were grown in YNB without amino acids containing 2%glucose for 2 days. The cells were harvested by centrifugation andresuspended in media comprising 100 g/L dextrose, 2 g/L MgSO₄ and 50 mMphosphate buffer at pH 6.5 for 5 additional days of growth. The cellsfrom 0.75 mL of each culture were harvested by centrifugation andanalyzed for their fatty acid composition. The fatty acid profile of 3transformants comprising the “control” vector and 5 transformantscomprising pTEF::ISC1 are shown below based on GC analysis (as describedin the General Methods). Fatty acids are identified as 16:0, 16:1, 18:0,18:1 (oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETA and EPA; and thecomposition of each is presented as a % of the total fatty acids.

TABLE 57 Lipid Composition In Yarrowia strain M4 Engineered ToOverexpress S. cerevisiae ISC1 Total Fatty Acids Strain 16:0 16:1 18:018:1 18:2 GLA DGLA ARA ETA EPA M4 + “control” 14.7 7.2 2.1 13.5 8.7 21.88.9 0.9 4.1 9.3 M4 + pTEF::ISC1 13.5 8.5 1.7 15.6 8.1 21.3 7.5 0.7 3.910.7

Expression of the S. cerevisiae ISC1 gene improved the percent EPA from9.3% in the “control” strain to 10.7% (“M4+pTEF::ISC1”), representing a14.5% increase.

Example 26 Generation of Yarrowia lipolytica Acyltransferase Knockouts

The present Example describes the creation of single, double and tripleknockout strains of Yarrowia lipolytica that were disrupted in eitherPDAT, DGAT2, DGAT1, PDAT and DGAT2, PDAT and DGAT1, DGAT1 and DGAT2, orPDAT, DGAT1 and DGAT2 genes. Disruption of the gene(s) in each of theknock-out strains was confirmed and analysis of each of the disruptionson fatty acid content and composition was determined by GC analysis oftotal lipids in Example 27.

Targeted Disruption of the Yarrowia lipolytica DGAT2 Gene

Targeted disruption of the DGAT2 gene in Y. lipolytica ATCC #90812 wascarried out by homologous recombination-mediated replacement of theendogenous DGAT2 gene with a targeting cassette designated as plasmidpY21DGAT2. pY21DGAT2 was derived from plasmid pY20 (FIG. 22D; SEQ IDNO:208). Specifically, pY21DGAT2 was created by inserting a 570 bp HindIII/Eco RI fragment into similarly linearized pY20. The 570 bp DNAfragment contained (in 5′ to 3′ orientation): 3′ homologous sequencefrom position +1090 to +1464 (of the coding sequence (ORF) in SEQ IDNO:130), a Bgl II restriction site and 5′ homologous sequence fromposition +906 to +1089 (of the coding sequence (ORF) shown in SEQ IDNO:130). The fragment was prepared by PCR amplification using two pairsof PCR primers, P95 and P96 (SEQ ID NOs:444 and 445), and P97 and P98(SEQ ID NOs:446 and 447), respectively.

pY21DGAT2 was linearized by Bgl II restriction digestion and transformedinto mid-log phase Y. lipolytica ATCC #90812 cells, according to theGeneral Methods. The cells were plated onto YPD hygromycin selectionplates and maintained at 30° C. for 2 to 3 days.

Fourteen Y. lipolytica ATCC #90812 hygromycin-resistant colonies wereisolated and screened for targeted disruption by PCR. One set of PCRprimers (P115 and P116 [SEQ ID NOs:448 and 449]) was designed to amplifya specific junction fragment following homologous recombination. Anotherpair of PCR primers (P115 and P112 [SEQ ID NO:450]) was designed todetect the native gene.

Two of the 14 hygromycin-resistant colonies of ATCC #90812 strains werepositive for the junction fragment and negative for the native fragment.Thus, targeted integration was confirmed in these 2 strains, one ofwhich was designated as “S-D2”.

Targeted Disruption of the Yarrowia lipolytica PDAT Gene

Targeted disruption of the PDAT gene in Y. lipolytica ATCC #90812 wascarried out by homologous recombination-mediated replacement of theendogenous PDAT gene with a targeting cassette designated as pLV13 (FIG.22E; SEQ ID NO:209). pLV13 was derived from plasmid pY20 (FIG. 22D; SEQID NO:208). Specifically, the hygromycin resistant gene of pY20 wasreplaced with the Yarrowia Ura3 gene to create plasmid pLV5. Then,pLV13. was created by inserting a 992 bp Bam HI/Eco RI fragment intosimilarly linearized pLV5. The 992 bp DNA fragment contained (in 5′ to3′ orientation): 3′ homologous sequence from position +877 to +1371 (ofthe coding sequence (ORF) in SEQ ID NO:117), a Bgl II restriction siteand 5′ homologous sequence from position +390 to +876 (of the codingsequence (ORF) in SEQ ID NO:117). The fragment was prepared by PCRamplification using PCR primers P39 and P41 (SEQ ID NOs:451 and 452) andP40 and P42 (SEQ ID NOs:453 and 454), respectively.

pLV13 was linearized by Bgl II restriction digestion and was transformedinto mid-log phase Y. lipolytica ATCC #90812 cells, according to theGeneral Methods. The cells were plated onto Bio101 DOB/CSM-Ura selectionplates and maintained at 30° C. for 2 to 3 days.

Ten Y. lipolytica ATCC #90812 colonies were isolated and screened fortargeted disruption by PCR. One set of PCR primers (P51 and P52 [SEQ IDNOs:455 and 456]) was designed to amplify the targeting cassette.Another set of PCR primers (P37 and P38 [SEQ ID NOs:457 and 458]) wasdesigned to detect the native gene. Ten of the ten strains were positivefor the junction fragment and 3 of the 10 strains were negative for thenative fragment, thus confirming successful targeted integration inthese 3 strains. One of these strains was designated as “S-P”.

Targeted Disruption of the Yarrowia lipolytica DGAT1 Gene

The full-length YI DGAT1 ORF was cloned by PCR using degenerate PCRprimers P201 and P203 (SEQ ID NOs:459 and 460, respectively) and Y.lipolytica ATCC #76982 genomic DNA as template. The degenerate primerswere required, since the nucleotide sequence encoding YI DGAT1 was notknown.

The PCR was carried out in a RoboCycler Gradient 40 PCR machine, withamplification carried out as follows: initial denaturation at 95° C. for1 min, followed by 30 cycles of denaturation at 95° C. for 30 sec,annealing at 55° C. for 1 min, and elongation at 72° C. for 1 min. Afinal elongation cycle at 72° C. for 10 min was carried out, followed byreaction termination at 4° C. The expected PCR product (ca. 1.6 kB) wasdetected by agarose gel electrophoresis, isolated, purified, cloned intothe TOPO® cloning vector (Invitrogen), and partially sequenced toconfirm its identity.

Targeted disruption of the putative DGAT1 gene in Y. lipolytica ATCC#90812 was carried out by homologous recombination-mediated replacementof the endogenous DGAT1 gene with a targeting cassette (using themethodology described above for DGAT2). Specifically, the 1.6 kBisolated YI DGAT1 ORF (SEQ ID NO:122) was used as a PCR templatemolecule to construct a YI DGAT1 targeting cassette consisting of: 5′homologous YI DGAT1 sequence (amplified with primers P214 and P215 (SEQID NOs:461 and 462)), the Yarrowia Leucine 2 (Leu2; GenBank AccessionNo. AAA35244) gene, and 3′ homologous YI DGAT1 sequence (amplified withprimers P216 and P217 (SEQ ID NOs:463 and 464)). Following amplificationof each individual portion of the targeting cassette with Pfu Ultrapolymerase (Stratagene, Catalog #600630) and the thermocycler conditionsdescribed above, each fragment was purified. The three correct-sized,purified fragments were mixed together as template molecules for asecond PCR reaction using PCR primers P214 and P219 (SEQ ID NO:465) toobtain the YI DGAT1 disruption cassette.

The targeting cassette was gel purified and used to transform mid-logphase wildtype Y. lipolytica (ATCC #90812). Transformation was performedas described in the General Methods. Transformants were plated ontoBio101 DOB/CSM-Leu selection plates and maintained at 30° C. for 2 to 3days. Several leucine prototrophs were screened by PCR to confirm thetargeted DGAT1 disruption. Specifically, one set of PCR primers (P226and P227 [SEQ ID NOs:466 and 467]) was designed to amplify a junctionbetween the disruption cassette and native target gene. Another set ofPCR primers (P214 and P217 [SEQ ID NOs:461 and 464]) was designed todetect the native gene.

All of the leucine prototroph colonies were positive for the junctionfragment and negative for the native fragment. Thus, targetedintegration was confirmed in these strains, one of which was designatedas “S-D1”.

Creation of Yarrowia lipolytica Double and Triple Knockout StrainsContaining Disruptions in PDAT and/or DGAT2 and/or DGAT1 Genes

The Y. lipolytica ATCC #90812 hygromycin-resistant “S-D2” mutant(containing the DGAT2 disruption) was transformed with plasmid pLV13(containing the PDAT disruption) and transformants were screened by PCR,as described for the single PDAT disruption. Two of twelve transformantswere confirmed to be disrupted in both the DGAT2 and PDAT genes. One ofthese strains was designated as “S-D2-P”.

Similarly, strains with double knockouts in DGAT1 and PDAT (“S-D1-P”),in DGAT2 and DGAT1 (“S-D2-D1”), and triple knockouts in DGAT2, DGAT1 andPDAT (“S-D2-D1-P”) were made.

Example 27 Yarrowia lipolytica Acyltransferase Knockouts Decrease LipidContent and Increase Percent PUFAs

The present Example analyzes the affect of single and/or double and/ortriple acyltransferase knockouts in wildtype Yarrowia lipolytica andstrains of Y. lipolytica that had been previously engineered to produceEPA, as measured by changes in fatty acid content and composition. It iscontemplated that a Y. lipolytica host strain engineered to produce DHAvia either the Δ6 desaturase/Δ6 elongase pathway or the Δ9 elongase/Δ8desaturase pathway could demonstrate increased DHA biosynthesis andaccumulation, if similar manipulations to the host's nativeacyltransferases were created (e.g., within strain Y3000).

TAG Content is Decreased in Y. lipolytica ATCC #90812 withAcyltransferase Disruptions

First, TAG content was compared in wildtype and mutant Y. lipolyticaATCC #90812 containing: (1) single disruptions in PDAT, DGAT2 and DGAT1;(2) double disruptions in PDAT and DGAT2, DGAT1 and PDAT, and DGAT1 andDGAT2; and (3) triple disruptions in PDAT, DGAT2 and DGAT1.

Specifically, one loopful of cells from plates containing wildtype andmutant Y. lipolytica ATCC #90812 (i.e., strains S-D1, S-D2, S-P,S-D1-D2, S-D1-P, S-D2-P, and S-D1-D2-P) were each individuallyinoculated into 3 mL YPD medium and grown overnight on a shaker (300rpm) at 30° C. The cells were harvested and washed once in 0.9% NaCl andresuspended in 50 mL of HGM. Cells were then grown on a shaker for 48hrs. Cells were washed in water and the cell pellet was lyophilized.Twenty (20) mg of dry cell weight was used for total fatty acid by GCanalysis and the oil fraction following TLC (infra) and GC analysis.

The methodology used for TLC is described below in the following fivesteps: (1) The internal standard of 15:0 fatty acid (10 μl of 10 mg/mL)was added to 2 to 3 mg dry cell mass, followed by extraction of thetotal lipid using a methanol/chloroform method. (2) Extracted lipid (50μl) was blotted across a light pencil line drawn approximately 1 inchfrom the bottom of a 5×20 cm silica gel 60 plate, using 25-50 μlmicropipettes. (3) The TLC plate was then dried under N₂ and wasinserted into a tank containing about ˜100 mL 80:20:1 hexane:ethylether:acetic acid solvent. (4) After separation of bands, a vapor ofiodine was blown over one side of the plate to identify the bands. Thispermitted samples on the other side of the plate to be scraped using arazor blade for further analysis. (5) Basic transesterification of thescraped samples and GC analysis was performed, as described in theGeneral Methods.

GC results are shown below in Table 58. Cultures are described as the“S” strain (wildtype), “S-P” (PDAT knockout), “S-D1” (DGAT1 knockout),“S-D2” (DGAT2 knockout), “S-D1-D2” (DGAT1 and DGAT2 knockout), “S-P-D1”(PDAT and DGAT1 knockout), “S-P-D2” (PDAT and DGAT2 knockout) and“S-P-D1-D2” (PDAT, DGAT1 and DGAT2 knockout). Abbreviations utilizedare: “WT”=wildtype; “FAs”=fatty acids; “dcw”=dry cell weight; and, “FAs% dcw, % WT”=FAs % dcw relative to the % in wildtype, wherein the “S”strain is wildtype.

TABLE 58 Lipid Content In Yarrowia ATCC #90812 Strains With Single,Double, Or Triple Disruptions In PDAT, DGAT2 And DGAT1 TAG Fraction FAsTotal Fatty Acids % FAs FAs % FAs dcw, Residual dcw, FAs, % dcw, % FAs,% % Strain DAG AT mg μg dcw WT μg dcw WT S D1, D2, P 32.0 797 15.9 100697 13.9 100 S-D1 D2, P 78.8 723 13.6 86 617 11.6 83 S-D2 D1, P 37.5 3296.4 40 227 4.4 32 S-P D1, D2 28.8 318 6.0 38 212 4.0 29 S-D1- P 64.6 2194.1 26 113 2.1 15 D2 S-D1-P D2 76.2 778 13.4 84 662 11.4 82 S-D2-P D131.2 228 4.3 27 122 2.3 17 S-D1- None 52.2 139 2.4 15 25 0.4 3 D2-P

The results in Table 58 indicate the relative contribution of the threeDAG ATs to oil biosynthesis. DGAT2 contributes the most, while PDAT andDGAT1 contribute equally but less than DGAT2. The residual oil contentca. 3% in the triple knockout strain may be the contribution of Yarrowialipolytica's acyl-CoA:sterol-acyltransferase enzyme, encoded by ARE2(SEQ ID NOs:119 and 120).

TAG Content is Decreased and Percent EPA is Increased in Yarrowialipolytica Strain EU with a Disrupted DGAT2 Gene

After examining the affect of various acyltransferase knockouts inwildtype Y. lipolytica ATCC #90812 (supra), TAG content and fatty acidcomposition was then studied in DGAT2 knockout strains of the EU strain(i.e., engineered to produce 10% EPA; see Example 13).

Specifically, the DGAT2 gene in strain EU was disrupted as described forthe S strain (ATCC #90812) in Example 26. The DGAT2-disrupted strain wasdesignated EU-D2. EU and EU-D2 strains were harvested and analyzedfollowing growth according to two different conditions. In the conditionreferred to in the Table below as “3 mL”, cells were grown for 1 day in3 mL MM medium, washed and then grown for 3 days in 3 mL HGM.Alternatively, in the condition referred to in the Table below as “51mL”, cells were grown for 1 day in 51 mL MM medium, washed and thengrown for 3 days in 51 mL HGM. The fatty acid compositions ofphosphatidylcholine (PC), phosphatidyletanolamine (PE), andtriacylglycerol (TAG or oil) were determined in the extracts of 51 mLcultures following TLC separation (“Fraction”).

GC results are shown below in Table 59. Cultures are described as the“EU” strain (wildtype) and the “EU-D2” strain (DGAT2 knockout). Fattyacids are identified as 16:0, 16:1, 18:0, 18:1 (oleic acid), 18:2 (LA),GLA, DGLA, ARA, ETA and EPA; and the composition of each is presented asa % of the total fatty acids.

TABLE 59 Lipid Content And Composition In Yarrowia Strain EU WithDisruption In DGAT2 Strain & TFAs % % % % % % % % % % % Growth Fractiondcw 16:0 16:1 18:0 18:1 18:2 GLA DGLA ARA ETA EPA EU, Total 19 10 2 1612 19 6 0 3 10 3 mL EU-D2, Total 17 10 1 6 7 24 5 0 6 19 3 mL EU, Total37 18 11 3 19 31 5 1 1 4 51 mL PC 2 12 9 1 8 43 7 3 5 4 PE 1 24 14 0 1437 5 0 0 1 TAG 34 18 12 3 21 29 5 1 1 4 EU-D2, Total 18 18 8 1 5 7 25 55 20 51 mL PC 1 18 6 1 2 4 26 5 11 22 PE 1 25 7 0 2 5 14 2 3 8 TAG 15 169 1 6 5 26 6 5 21

The results show that the DGAT2 knockout resulted in doubling of the %EPA (of total fatty acids) and halving of the lipid content (as % dcw).Furthermore, almost all of the changes observed in the lipid content aredue to changes in the TAG fraction. The lower than expected % EPA in the51 mL culture of strain EU is likely due to instability.

TAG Content is Decreased and Percent EPA is Increased in Yarrowialipolytica Strain MU with Disrupted Acyltransferase Genes

Finally, based on the increased % EPA and reduced lipid contentresulting from a single DGAT2 knockout in strain EU-D2, TAG content andfatty acid composition was then studied in various acyltransferaseknockout strains of strain MU (engineered to produce 14% EPA; seeExample 15). Specifically, single disruptions in PDAT, DGAT2 and DGAT1and double disruptions in PDAT and DGAT2 were created in strain MU.Lipid content and composition was compared in each of these strains,following growth in 4 different growth conditions.

More specifically, single disruptions in PDAT, DGAT2, DGAT1 were createdin strain MU, using the methodology described in Example 26 (with theexception that selection for the DGAT1 disruption relied on the URA3gene). This resulted in single knockout strains identified as “MU-D1”(disrupted in DGAT1), “MU-D2” (disrupted in DGAT2), and “MU-P”(disrupted in PDAT). Individual knockout strains were confirmed by PCR.Additionally, the MU-D2 strain was disrupted for the PDAT gene by thesame method and the disruption confirmed by PCR. The resulting doubleknockout strain was designated “MU-D2-P”.

The MU-D1, MU-D2, MU-P, and M-D2-P knockout strains were analyzed todetermine each knockout's effect on lipid content and composition, asdescribed below. Furthermore, the growth conditions promoting oleaginywere also explored to determine their effect on total lipid content.Thus, in total, four different experiments were conducted, identified as“Experiment A”, “Experiment B”, “Experiment C” and “Experiment E”.Specifically, three loops of cells from plates containing each strainabove was inoculated into MMU medium [3 mL for Experiments B and C; and50 mL for Experiments A and E] and grown in a shaker at 30° C. for 24hrs (for Experiments A, B and C) or 48 hrs (for Experiment E). Cellswere harvested, washed once in HGM, resuspended in either HGM medium (50mL for Experiments A and E; and 3 mL for Experiment B) or HGM mediumwith uracil (“HGMU”) (3 mL for Experiment C) and cultured as above for 4days. One aliquot (1 mL) was used for lipid analysis by GC as describedaccording to the General Methods, while a second aliquot was used fordetermining the culture OD at 600 nm. The remaining culture inExperiments A and E was harvested, washed once in water, and lyophilizedfor dry cell weight (dcw) determination. In contrast, the dcw inExperiments B and C were determined from their OD₆₀₀ using the equationshowing their relationship. The fatty acid compositions of each of thedifferent strains in Experiments A, B, C and E was also determined.

The results are shown in Table 60 below. Cultures are described as the“MU” strain (the parent EPA producing strain), “MU-P” (PDAT knockout),“MU-D1” (DGAT1 knockout), “MU-D2” (DGAT2.knockout) and “MU-D2-P” (DGAT2and PDAT knockouts). Abbreviations utilized are: “WT”=wildtype (i.e.,MU); “OD”=optical density; “dcw”=dry cell weight; “TFAs”=total fattyacids; and, “TFAs % dcw, % WT”=TFAs % dcw relative to the wild type(“MU”) strain. Fatty acids are identified as 16:0, 16:1, 18:0, 18:1(oleic acid), 18:2 (LA), GLA, DGLA, ARA, ETA and EPA; and thecomposition of each is presented as a % of the total fatty acids.

TABLE 60 Lipid Content And Composition In Yarrowia Strain MU WithVarious Acyltransferase Disruptions 1^(st) Phase 2^(nd) Phase TFAs %Residual Growth Growth dcw TFAs TFAs dcw, Expt Strain DAG AT ConditionCondition OD (mg) (μg) % dcw % WT A MU D1, D2, P 1 day, 4 days, 4.0 91374 20.1 100 A MU-D2 D1, P 50 mL 50 mL 3.1 75 160 10.4 52 A MU-D1 D2, PMMU HGM 4.3 104 217 10.2 51 A MU-P D1, D2 4.4 100 238 11.7 58 B MU D1,D2, P 1 day, 4 days, 5.9 118 581 24.1 100 B MU-D2 D1, P 3 mL 3 mL 4.6102 248 11.9 50 B MU-D1 D2, P MMU HGM 6.1 120 369 15.0 62 B MU-P D1, D26.4 124 443 17.5 72 C MU D1, D2, P 1 day, 4 days, 6.8 129 522 19.9 100 CMU-D2 D1, P 3 mL 3 mL 5.6 115 239 10.2 51 C MU-D1 D2, P MMU HGMU 6.9 129395 15.0 75 C MU-P D1, D2 7.1 131 448 16.8 84 E MU D1, D2, P 2 days, 4days, 4.6 89 314 17.3 100 E MU-D2 D1, P 50 mL 50 mL 2.8 62 109 8.5 49 EMU-P D2, P MM HGM 5.0 99 232 11.5 66 E MU-D2-P D1 4.2 98 98 4.9 28 % % %% % % % % % Expt Strain 16:0 16:1 18:0 18:1 18:2 GLA DGLA ARA ETA % EPAA MU 17 10 2 18 10 22 7 1 3 9.7 A MU-D2 16 12 0 8 9 23 7 0 8 17.4 AMU-D1 15 10 2 11 10 22 7 0 7 17.4 A MU-P 16 9 2 11 7 24 7 1 6 17.5 B MU17 9 3 18 10 22 8 1 3 9.1 B MU-D2 16 10 0 7 10 24 7 1 7 17.8 B MU-D1 189 3 14 11 20 7 1 5 12.0 B MU-P 15 8 3 16 10 25 6 1 4 11.9 C MU 16 10 213 11 21 10 1 4 12.6 C MU-D2 17 9 1 6 11 21 8 1 7 18.9 C MU-D1 15 9 2 1212 20 10 1 5 13.5 C MU-P 17 8 3 14 11 20 10 1 4 11.3 E MU 16 12 2 18 922 7 1 4 11.2 E MU-D2 14 12 1 6 8 25 6 0 7 20.0 E MU-P 16 10 2 14 7 24 71 5 15.8 E MU-D2-P 18 10 0 7 12 20 5 0 6 22.5

The data showed that the lipid content within the transformed cellsvaried according to the growth conditions. Furthermore, the contributionof each acyltransferase on lipid content also varied. Specifically, inExperiments B, C and E, DGAT2 contributed more to oil biosynthesis thaneither PDAT or DGAT1. In contrast, as demonstrated in Experiment A, asingle knockout in DGAT2, DGAT1 and PDAT resulted in approximatelyequivalent losses in lipid content (i.e., 48%, 49% and 42% loss,respectively [see “TFAs % dcw, % WT”]).

With respect to fatty acid composition, the data shows that knockout ofeach individual DAG AT gene resulted in lowered oil content andincreased % EPA. For example, the DGAT2 knockout resulted in about halfthe lipid content and ca. double the % EPA in total fatty acids (similarto the results observed in strain EU-D2, supra). Knockout of both DAGAT2and PDAT resulted in the least oil and the most % EPA.

On the basis of the results reported herein, it is contemplated thatdisruption of the native DGAT2 and/or DGAT1 and/or PDAT is a usefulmeans to substantially increase the % PUFAs in a strain of Yarrowialipolytica engineered to produce high concentrations of PUFAs, includingDHA (e.g., within strain Y3000). In fact, a disruption of the nativeDGAT2 gene in Y. lipolytica strain Y2214 (producing 14% ARA via the Δ9elongase/Δ8 desaturase pathway; the final genotype of this strain withrespect to wildtype Y. lipolytica ATCC #20362 was as follows: Aco2-,Lys5-, 2×GPAT::IgD9e::PEX20, 2×TEF::IgD9e::LIP1, FBAINm::IgD9e::OCT,2×FBAIN::D8SF::PEX16, GPD::D8SF::PEX16, GPAT::MAΔ5::PEX20,FBAIN::MAΔ5::PEX20, YAT1::I.D5S::LIP1, GPM/FBAIN::I.D5S::OCT,FBAIN::F.D12S::PEX20 and GPM/FBAIN::rELO2S::OCT) resulted in a 1.7 foldincrease in the percent ARA (data not shown).

1-5. (canceled)
 6. A recombinant oleaginous Yarrowia yeast cell for theproduction of docosahexaenoic acid comprising a background Yarrowia sp.comprising: a) at least one gene encoding Δ9 elongase; and b) at leastone gene encoding Δ8 desaturase; wherein the oleaginous Yarrowia yeastcell accumulates in excess of about 25% of its dry cell weight as oiland the oleaginous Yarrowia yeast cell produces at least about 5%docosahexaenoic acid of total lipids produced.
 7. A microbial oilproduced by a method comprising: a) culturing the recombinant oleaginousYarrowia yeast cell of claim 6 wherein a microbial oil comprisingdocosahexaenoic acid is produced; and b) optionally, recovering themicrobial oil of step (a).
 8. The microbial oil of claim 7 wherein theoil comprises at least one of the following selected from the groupconsisting ot: a) comprises at least about 5% docosahexaenoic acid andb) the oil is devoid of any γ-linoleic acid.
 9. The microbial oil ofclaim 7 blended with an oil comprising a fatty acid selected from thegroup consisting of linoleic acid, γ-linolenic acid, eicosadienoic acid,dihomo-γ-linolenic acid, arachidonic acid, α-linolenic acid, stearidonicacid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid,docosapentaenoic acid and docosahexaenoic acid.
 10. A food productcomprising an effective amount of the microbial oil of claim
 7. 11. Afood product according to claim 10 selected from the group consisting ofa food analogue, a meat product, a cereal product, a baked food, a snackfood and a dairy product.
 12. A product selected from the groupconsisting of a medical food, a dietary supplement, an infant formulaand a pharmaceutical, comprising an effective amount of the microbialoil of claim
 7. 13. An animal feed comprising an effective amount of themicrobial oil of claim
 7. 14. An animal feed according to claim 13wherein the animal feed is selected from the group consisting of a petfeed, a ruminant feed, a poultry feed, and an aquacultural feed.
 15. Ananimal feed comprising an effective amount of microbial oil andoptionally comprising a yeast biomass comprising the recombinantoleaginous Yarrowia yeast cell of claim
 6. 16. An animal feed accordingto claim 15 wherein the yeast biomass comprises feed nutrients selectedfrom the group consisting of proteins, lipids, carbohydrates, vitamins,minerals and nucleic acids.